Compositions and methods to treating hemoglobinopathies

ABSTRACT

Embodiments herein provide specially designed synthetic BCL11A-targeting microRNAs for RNA polymerase II expression, and methods of use to treat hemoglobinopathies such as sickle cell disease or thalassemia by increasing the expression levels of fetal hemoglobin levels.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation Application of U.S. application Ser.No. 16/841,259 filed Apr. 6, 2020, which is a Continuation Applicationof U.S. application Ser. No. 16/366,900 filed Mar. 27, 2019, now U.S.Pat. No. 10,662,429, issued May 26, 2020, which is a DivisionalApplication of U.S. application Ser. No. 15/306,527 filed Oct. 25, 2016,now U.S. Pat. No. 10,287,588, issued May 14, 2019, which is a 35 U.S.C.371 National Stage Application of International Application No.PCT/US2015/027527 filed Apr. 24, 2015, which designates the UnitedStates and which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/984,247, filed Apr. 25, 2014, and U.S.Provisional Application No. 62/066,783, filed Oct. 21, 2014 the contentsof each application are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.:5U01HL117720-03 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 26, 2020, isnamed 701039-075992-PCT_SL and is 81,309 bytes in size.

TECHNICAL FIELD

Embodiments disclosed herein relate to compositions and methods for thetreatment of hemoglobinopathies. More particularly, the embodimentsrelate to compositions and methods of increasing fetal hemoglobin in acell by selective knockdown of the endogenous BCL11A.

BACKGROUND

Hemoglobinopathies, including sickle cell disease/anemia (SCD) andthalassemia (THAL), are the most prevalent inherited monogenic disordersin the human. Approximately 5% of the world's population carries aglobin gene mutation. The World Health Organization estimates that eachyear about 300,000 infants are born with major hemoglobin disorders. SCDhas segregated to populations from sub-Saharan Africa, India, SaudiArabia, and Mediterranean countries, where up to 2% of all children areborn with the condition, due to the survival advantage against malarialtransmission conferred by a heterozygous sickle β-globin (βS) mutation(WHO Report on Sickle-cell anaemia—A59.9. Fifty-ninth World HealthAssembly—Provisional agenda item 114: United Nations; 2006:1-5). Due tohistoric and/or recent migration, increasing numbers of patientpopulations can now be found in developed countries, and public healthimplications of SCD are significant (Kauf et al., American Journal ofHematology. 2009; 84:323-327; Amendah et al., American Journal ofPreventive Medicine. 2010; 38:5550-556). In the United States ofAmerica, median survival of patients having a hemoglobinopathy wasestimated in 1994 to be 42 years for men and 48 years for women (Plattet al., New England Journal of Medicine. 1994; 330:1639-1644). At amolecular level, SCD was the first disease to be linked to a molecularalteration (Pauling et al., Science. 1949; 110:543-548). A singlenucleotide mutation results in glutamic acid to valine substitution byat position 6 of the β-globin protein. This modification results in thepolymerization of the molecule in deoxygenated conditions, andsubsequent “sickling” of the erythrocyte ultimately leading to anemia byhemolysis and acute and chronic vaso-occlusive and ischemiccomplications affecting multiple organs, including kidney, brain, lung,and others). Although preventive measures (including thechemoprophylactic agent hydroxyurea) have led to moderate reduction inthe burden of selected patient groups, at present, the only availablecurative therapy for SCD is allogeneic hematopoietic stem celltransplantation (HSCT) (Hsieh et al., New England Journal of Medicine.2009; 361:2309-2317; Hsieh et al., Blood; Electronic pre-publicationJun. 31, 2011). HSCT has unfortunately been associated in the SCD andTHAL setting with significant mortality and morbidity, which is due inpart to pre-HSCT transfusion-related iron overload, graft-versus-hostdisease, and high doses of chemotherapy/radiation required forpre-transplant conditioning of the host, among others.

New molecular therapies are being developed. For example, U.S. Pat. No.8,383,604 describes that the BCL11A as a key regulator of the globingenes during development. In particular, BCL11A promotes thetransitional switch from the expression of fetal hemoglobin genes to theexpression of adult hemoglobin genes during fetal development.Suppression of BCL11A reduces this transitional switch and maintains asignificantly higher expression of the fetal hemoglobin genes post fetaldevelopment. The higher amount of fetal hemoglobin genes expressedameliorates the symptoms associated with various hemoglobinopathies.

SUMMARY

In particular illustrative embodiments, the present invention provides,in part, improved compositions and methods for achieving gene therapy inhematopoietic cells and hematopoietic precursor cells, includingerythrocytes, erythroid progenitors, and embryonic stem cells. Theinvention further provides improved gene therapy methods for treatinghematopoietic-related disorders.

The goal is to efficiently knock-down BCL11A in cells derived fromtransduced, engraftable hematopoietic stem cells. Success at inductionof γ-globin and thus simultaneous increase in HbF and reduction inmutant HbS depends on the quantitative reduction of BCL11A transcriptand protein. The inventors have embedded a BCL11A shRNA in a mir223loop. This approach allows the BCL11A shRNA to be transcribed viapolymerase II (PolII) promoters instead of the polymerase III promoters.This allows exploitation of the microRNA-biogenesis pathway to generatesiRNAs that target BCL11A expression in engraftable HSCs. Lentiviraltransgenes are engineered to express shRNAs that mimic primary microRNAs(pri-miRNAs) and are sequentially processed by the endogenousMicroprocessor and Dicer complexes to generate small interfering RNAs(siRNAs) with sequence complementarity to the BCL11A messenger RNA(mRNA).

In one aspect, compositions and methods are provided that efficientlyknock-down BCL11A in cells derived from transduced, engraftablehematopoietic stem cells. In one embodiment, a quantitative reduction ofBCL11A transcript and protein induces γ-globin production, and thussimultaneous increase in HbF and reduction in mutant HbS. In aparticular embodiment, a BCL11A shRNA is embedded in a mir223 loop. Inparticular embodiments, a lentivirus is engineered to express shRNAsthat mimic pri-miRNAs that are sequentially processed by the endogenousMicroprocessor and Dicer complexes to generate siRNAs with sequencecomplementarity to the BCL11A mRNA.

Accordingly, in various illustrative embodiments, the presentspecification provides, in part, a synthetic BCL11A microRNA comprisinga first BCL11A segment, a loop segment, and a second BCL11A segmentarranged in tandem in a 5′ to 3′ direction, wherein the loop segment isbetween and directly linked to the first and second BCL11A segments, andwherein the second BCL11A segment is complementary to the first BCL11Asegment such that the first and second BCL11A segments base pair to forma hairpin loop with the loop segment forming the loop portion of thehairpin loop thus formed.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first and second BCL11A segments are about 18 to 25nucleotides long. The first BCL11A segment is derived from a BCL11Asequence and gives rise to the passenger strand during shRNA processingto a duplex siRNA and the second BCL11A segment is complementary tofirst BCL11A segment, wherein the second BCL11A segment gives rise tothe guide strand that is incorporated into the RNA InterferenceSpecificity Complex (RISC) for RNA interference or BCL11A genesilencing.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first and second BCL11A segments contain sequences that arederived from BCL11A mRNA sequence.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment starts with a -GCGC- at the 5′ end andthe second BCL11A segment ends with a -GCGC- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment further consist a -GCGC- at the 5′ endand the second BCL11A segment ends with a -GCGC- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment starts with a -GCGA-, -TCTG-, or -TG-at the 5′ end and the second BCL11A segment is complementary to firstBCL11A segment.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment further consist a -GCGA-, -TCTG-, or-TG- at the 5′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the second BCL11A segment ends with a -TTTT- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA comprise a nucleotide sequenceselected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA comprises a nucleotide sequence ora segment therefrom described in this disclosure.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA consists of a nucleotide sequenceselected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA consist essentially of anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment is selected from the group consistingof CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; derived from BCL11A miR1oligo described herein), CCAGAGGATGACGATTGTTTA (SEQ. ID. NO: 47; derivedfrom BCL11A miR2 oligo described herein), TCGGAGACTCCAGACAATCGC (SEQ.ID. NO: 48; derived from BCL11A E3 oligo or shRNA1 or E3 describedherein), CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 orB5 described herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50; derivedfrom shRNA4 or B11 described herein), TTCTCTTGCAACACGCACAGA (SEQ. ID.NO: 51; derived from BCL11A D8 oligo or shRNA3 or D8 described herein),GATCGAGTGTTGAATAATGAT (SEQ. ID. NO: 52; derived from shRNA5 or 50D12 o1D12 described herein), CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53; derivedfrom shRNA5 or 50A5 described herein), CACTGTCCACAGGAGAAGCCA (SEQ. ID.NO: 54; derived from shRNA7 or 50B11 described herein),ACAGTACCCTGGAGAAACACA (SEQ. ID. NO: 55; derived from BCL11A XLC4, shRNA8and 50C4 described herein), CAACAAGATGAAGAGCACCAA (SEQ. ID. NO: 56;derived from BCL11A Non-targeting oligos described herein),gcgcCGCACAGAACACTCATG (SEQ. ID. NO: 57; derived from miR1G5 oligodescribed herein), GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58; derived fromE3G5 or E3 mod oligo or shRNA1 mod described herein),gcgcCCTCCAGGCAGCTCAAA (SEQ. ID. NO: 59; derived from B5G5 or shRNA2 moddescribed herein); gcgcTCAGGACTAGGTGCAGA (SEQ. ID. NO: 60; derived fromB11G5 or shRNA4 mod described herein); gcgcGATCGAGTGTTGAATAA (SEQ. ID.NO: 61; derived from 50D12G5, D12G4 or shRNA5 mod described herein);gcgcCAGTACCCTGGAGAAAC (SEQ. ID. NO: 62; derived from 50A5G5 or shRNA6mod described herein); gcgcCACTGTCCACAGGAGAA (SEQ. ID. NO: 63; derivedfrom 50B11G5 or shRNA7 mod described herein); GCGCTTCTCTTGCAACACGCA(SEQ. ID. NO: 64; derived from BCL11A D8G5 or D8 mod or shRNA3 moddescribed herein), GCGCACAGTACCCTGGAGAAA (SEQ. ID. NO: 65; derived fromBCL11A C4G5, or C4 mod or shRNA8 mod described herein),CGCACAGAACACTCATGGATT (SEQ. ID. NO: 66; derived from BCL11A D12G5-2described herein), and ACGCTCGCACAGAACACTCATGGATT (SEQ. ID. NO: 67;derived from BCL11A D12G5-2 described herein).

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the loop segment is derived from a microRNA. In one embodiment,the microRNA is a hematopoietic specific microRNA. For examples,miR-142, miR-155, miR-181 and miR-223.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the microRNA is miR223.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the loop segment is ctccatgtggtagag (SEQ ID NO:68).

Accordingly, in one aspect, the present specification provides anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or asynthetic BCL11A microRNA described herein.

Accordingly, in one aspect, the present specification provides acomposition comprising at least one nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.

Accordingly, in one aspect, the present specification provides acomposition comprising at least a vector comprising a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11AmicroRNA described herein.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:1.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:2.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:3.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:4.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:5.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:6.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:7.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:8.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:9.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:10.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:13.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:14.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:15.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:16.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:17.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:18.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:25.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:26.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:27.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:28.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:29.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:30.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:31.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:32.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:33.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:34.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:35.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:36.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:37.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:38.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:39.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:40.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:41.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:42.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:43.

In one embodiment of any isolated nucleic acid molecule described, themolecule comprises the nucleotide sequence of SEQ ID NO:44.

In one aspect, the present specification provides a vector comprising atleast one nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44 or asynthetic BCL11A microRNA described herein.

In one embodiment of any vector described, the vector further comprisesa spleen focus-forming virus promoter, a tetracycline-induciblepromoter, or a β-globin locus control region and a β-globin promoter.The promoter provide for targeted expression of the nucleic acidmolecule therein or the synthetic BCL11A microRNA therein.

In one aspect, the present specification provides a host cell comprisinga vector which comprises at least one nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.

In one embodiment of any host cell described herein, the host cell is anembryonic stem cell, a somatic stem cell, a progenitor cell, a bonemarrow cell, a hematopoietic stem cell, or a hematopoietic progenitorcell. In one embodiment, the host cell is isolated from a subject. Inone embodiment, the host cell is isolated from a subject who has beendiagnosed with a hemoglobinopathy. Diagnosis can be made by any methodknown in the art. For example, by genetic testing, by RT-PCR, and byblood cytology.

In one embodiment of any host cell described herein, the host cell is anerythrocyte.

In one aspect, the present specification provides a host cell comprisinga vector or a bacterium which comprises at least one nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11AmicroRNA described herein.

In one aspect, the present specification provides a host cell comprisinga virus which comprises at least one nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.

In one embodiment of any virus or vector described herein, the virus isa lentivirus.

In one embodiment of any vector or virus described herein, thelentivirus is selected from the group consisting of: humanimmunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equineinfectious anemia virus (EIAV), feline immunodeficiency virus (FIV),bovine immune deficiency virus (BIV), and simian immunodeficiency virus(SIV).

Accordingly, one aspect of, the present specification provides methodsfor increasing fetal hemoglobin levels expressed by a cell, comprisingthe steps of contacting an embryonic stem cell, a somatic stem cell, aprogenitor cell, a bone marrow cell, a hematopoietic stem cell, or ahematopoietic progenitor cell with an effective amount of a compositiondescribed herein or an effective amount of at least isolated nucleicacid molecule described herein, whereby fetal hemoglobin expression isincreased in the cell, or its progeny, relative to the cell prior tosuch contacting. In some embodiments, the composition comprises at leastone vector or cell comprising at least one nucleic acid moleculecomprising the nucleotide sequence selected from the group consisting ofSEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA describedherein. In one embodiment, the method further comprises providing asample of stem or progenitor cells for the contacting. In oneembodiment, the sample of cells comprises CD34+ selected cells. In oneembodiment, the composition comprises a mixture of the nucleotidesequences selected from the group consisting of SEQ ID NOS:1-10,13-18,25-44. For example, the composition has 2-5 different nucleotidesequences selected from the group consisting of SEQ ID NOS:1-10,13-18,25-44. For example, the composition comprises SEQ ID NOS: 34, 37, 39, 41and 43.

In one aspect, the present specification provides methods of treating,or reducing a risk of developing, a hemoglobinopathy, e.g., SCD andTHAL, in a subject. The methods can include selective knockdown of theBCL11A gene in the hematopoietic stem cells of subjects or individuals.These subjects are at risk of developing, a hemoglobinopathy.

In one embodiment of any method described, the selective knockdown ofthe BCL11A gene in the hematopoietic stem cells comprises using anisolated nucleic acid molecule comprising a nucleotide sequence of SEQID NOS:1-10,13-18, 25-44 or using a vector (e.g. a viral vector)comprising a nucleic acid molecule comprising any one of the nucleotidesequence of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNAdescribed herein.

In one embodiment of any method described, the selective knockdown ofthe BCL11A gene in the hematopoietic stem cells comprises contacting thehematopoietic stem cells with a composition which comprises at least anisolated nucleic acid molecule comprising the nucleotide sequence of SEQID NOS:1-10,13-18, 25-44, or with a composition which comprises at leasta vector (e.g. a viral vector) comprising a nucleic acid moleculecomprising any one of the nucleotide sequence of SEQ ID NOS:1-10,13-18,25-44, or a synthetic BCL11A microRNA described herein. In oneembodiment, the hematopoietic stem cells are isolated prior thecontacting.

In one embodiment of any method described, the selective knockdown ofthe BCL11A gene in the hematopoietic stem cells occurs in vivo, invitro, or ex vivo. In a further embodiment, the hematopoietic progenitorcell being targeted for selective knockdown is of the erythroid lineage.

In one embodiment of any method described, the contacting of thehematopoietic stem cells with any of the composition described hereinoccurs in vivo, in vitro, or ex vivo. In a further embodiment, thehematopoietic progenitor cell being contacted is of the erythroidlineage.

In one embodiment of any method described, the contacting of thehematopoietic stem cells with any of the composition described hereinoccurs in vivo, in vitro, or ex vivo.

In other embodiments of any method described, selective knockdown of theBCL11A gene occurs in an embryonic stem cell, a somatic stem cell, aprogenitor cell, a bone marrow cell in addition to a hematopoietic stemcell, or a hematopoietic progenitor cell. In one embodiment, anembryonic stem cell, a somatic stem cell, a progenitor cell, or a bonemarrow cell is contacted with the described composition. The embryonicstem cell, the somatic stem cell, the progenitor cell, or the bonemarrow cell is isolated prior the contacting. In one embodiment, thecontacting of the embryonic stem cell, the somatic stem cell, theprogenitor cell, or the bone marrow cell with any of the compositiondescribed herein occurs in vivo, in vitro, or ex vivo.

In other embodiments of any method described, the hematopoietic stemcells are collected from peripheral blood, cord blood, chorionic villi,amniotic fluid, placental blood, or bone marrow.

In other embodiments of any method described, the embryonic stem cell,somatic stem cell, progenitor cell, or bone marrow cell is collectedfrom peripheral blood, cord blood, chorionic villi, amniotic fluid,placental blood, or bone marrow.

In one aspect, the present specification provides a method of treating,or reducing a risk of developing, a hemoglobinopathy in a subject, themethod comprising: administering to the subject a therapeuticallyeffective amount of one or more isolated nucleic acid molecule describedherein, a virus or a vector described herein, or a cell describedherein, thereby treating, or reducing the risk of developing, thehemoglobinopathy in the subject, wherein the virus, the vector or cellcomprises at least one nucleic acid molecule comprising the nucleotidesequence selected from the group consisting of SEQ ID NOS:1-10,13-18,25-44, or a synthetic BCL11A microRNA described herein. For example, theeffective amount of one or more isolated nucleic acid molecule describedherein, a virus or a vector described herein, or a cell described hereinare injected directly into the bone marrow of the subject.

In one aspect, the present specification provides a method of treating,or reducing a risk of developing, a hemoglobinopathy in a subject, themethod comprising contacting a population of hematopoietic stem cells invitro or ex vivo with a composition described herein or with at leastone or more isolated nucleic acid molecule described herein, a virus ora vector described herein, and implanting or administering the contactedhematopoietic stem cells or the progeny cells thereof to the subject. Inone embodiment, the contacted hematopoietic stem cells or the progenycells engrafts in the subject. In one embodiment, the contactedhematopoietic stem cells or the progeny cells thereof are implanted withprostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to promotethe engraftments of the contacted cells.

In one aspect, the present specification provides a method of treating,or reducing a risk of developing, a hemoglobinopathy in a subject, themethod comprising expressing at least one synthetic BCL11A microRNAdescribed herein in an embryonic stem cell, a somatic stem cell, aprogenitor cell, a bone marrow cell, a hematopoietic stem cell, or ahematopoietic progenitor cell of the subject wherein the expression isex vivo or in vitro, and implanting or administering the cell into thesubject.

In one aspect, the present specification provides a method forincreasing fetal hemoglobin levels expressed by a cell, the methodcomprising expressing at least one synthetic BCL11A microRNA describedherein in an embryonic stem cell, a somatic stem cell, a progenitorcell, a bone marrow cell, a hematopoietic stem cell, or a hematopoieticprogenitor cell of a subject wherein the expression is ex vivo or invitro or in vivo. In one embodiment, the expression is by contacting thecells with an effective amount of a composition described herein or aneffective amount of at least isolated nucleic acid molecule describedherein.

In one aspect, the present specification provides a method fordecreasing BCL11A levels expressed by a cell, the method comprisingexpressing at least one synthetic BCL11A microRNA described herein in anembryonic stem cell, a somatic stem cell, a progenitor cell, a bonemarrow cell, a hematopoietic stem cell, or a hematopoietic progenitorcell of a subject wherein the expression is ex vivo or in vitro or invivo. In one embodiment, the expression comprises the steps ofcontacting an embryonic stem cell, a somatic stem cell, a progenitorcell, a bone marrow cell, a hematopoietic stem cell, or a hematopoieticprogenitor cell with an effective amount of a composition describedherein or an effective amount of at least isolated nucleic acid moleculedescribed herein, whereby fetal hemoglobin expression is increased inthe cell, or its progeny, relative to the cell prior to such contacting.In some embodiments, the composition comprises at least one vector orcell comprising at least one nucleic acid molecule comprising thenucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.

In a further embodiment of any methods described herein, thehematopoietic stem cell or hematopoietic progenitor cell being contactedis of the erythroid lineage.

In one embodiment of any methods described herein, the hematopoieticstem cell or hematopoietic progenitor cell is collected from peripheralblood, cord blood, chorionic villi, amniotic fluid, placental blood, orbone marrow.

In a further embodiment of any methods described herein, the recipientsubject is treated with chemotherapy and/or radiation prior toimplantation of the contacted or transfected cells.

In one embodiment, the chemotherapy and/or radiation is to reduceendogenous stem cells to facilitate engraftment of the implanted cells.

In one aspect, the present specification provides a method of treating,or reducing a risk of developing a hemoglobinopathy in a subject, themethod comprising providing hematopoietic stem cells from the subject,contacting the hematopoietic stem cells in vitro or ex vivo with acomposition described herein or with at least one or more isolatednucleic acid molecule described herein, a virus or a vector describedherein, and implanting or re-administering the contacted hematopoieticstem cells back into the same subject. In one embodiment, the contactedhematopoietic stem cells or the progeny cells engrafts in the subject.

In one aspect of any method, the contacted hematopoietic stem cells,embryonic stem cells, somatic stem cells, progenitor cells, bone marrowcells, or the progeny cells thereof are treated ex vivo withprostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to promotesubsequent engraftment in a recipient subject.

In one aspect of any method, the population of hematopoietic stem cellsor host cells is obtained from a subject at risk of developing ahemoglobinopathy or has been diagnose with a hemoglobinopathy.

In one aspect of any method, the population of hematopoietic stem cellsis autologous or allogeneic to the subject.

In one aspect of any method, the population of hematopoietic stem cellsor host cells is ex vivo expanded in culture prior to contacting with acomposition described herein or with at least one or more isolatednucleic acid molecule described herein, a virus or a vector describedherein.

In one aspect of any method, the population of hematopoietic stem cellsor host cells is ex vivo expanded in culture after to contacting with acomposition described herein or with at least one or more isolatednucleic acid molecule described herein, a virus or a vector describedherein.

In one aspect of any method, the contacted population of hematopoieticstem cells or host cells is pre-differentiated ex vivo in culture priorto implanting into a subject.

In one aspect of any method, the contacted hematopoietic stem cells areexpanded in vitro or ex vivo prior to administering into the subject. Inone aspect of any method, the contacted hematopoietic stem cells arecryopreserved prior to administering into the subject. In another aspectof any method, the contacted hematopoietic stem cells are expanded invitro or ex vivo and cryopreserved prior to administering into thesubject. In another aspect of any method, the contacted hematopoieticstem cells are expanded in vitro or ex vivo after cryopreservation priorto administering into the subject.

In one aspect of any method, the subject is a human. In one aspect ofany method, the subject is diagnosed with a hemoglobinopathy.

In one aspect of any method, the method further comprises of selecting asubject diagnosed with a hemoglobinopathy or a subject at risk ofdeveloping a hemoglobinopathy.

In one aspect of any method, the hemoglobinopathy is sickle cell disease(SCD) or thalassemia (THAL). For example, β-thalassemias.

In one aspect of the method, the method further comprising administeringto the subject a therapy comprising oxygen, hydroxyurea, folic acid, ora blood transfusion.

In one aspect, the present specification provides a method of treating,or reducing a risk of developing, a hemoglobinopathy in a subject, themethod comprising expressing in vivo at least one synthetic BCL11AmicroRNA described herein in the subject.

In one aspect of any method, the in vivo expression occurs in anembryonic stem cell, a somatic stem cell, a progenitor cell, a bonemarrow cell, a hematopoietic stem cell, or a hematopoietic progenitorcell.

In one aspect of any method, the embryonic stem cell, somatic stem cell,progenitor cell, bone marrow cell, hematopoietic stem cell, orhematopoietic progenitor cell is autologous or allogeneic to thesubject.

In one aspect of any method, the embryonic stem cell, somatic stem cell,progenitor cell, bone marrow cell, hematopoietic stem cell, orhematopoietic progenitor cell expressing the at least one syntheticBCL11A microRNA described herein is expanded in vitro or ex vivo priorto administering into the subject. In a further embodiment, theprogenitor cell, bone marrow cell, hematopoietic stem cell andhematopoietic progenitor cell is of the erythroid lineage.

In one aspect of any method, the embryonic stem cell, somatic stem cell,progenitor cell, bone marrow cell, hematopoietic stem cell, orhematopoietic progenitor cell expressing the at least one syntheticBCL11A microRNA described herein is cryopreserved prior to administeringinto the subject.

In another aspect of any method, the embryonic stem cell, somatic stemcell, progenitor cell, bone marrow cell, hematopoietic stem cell, orhematopoietic progenitor cell expressing the at least one syntheticBCL11A microRNA described herein is expanded in vitro or ex vivo andcryopreserved prior to administering into the subject.

In another aspect of any method, the embryonic stem cell, somatic stemcell, progenitor cell, bone marrow cell, hematopoietic stem cell, orhematopoietic progenitor cell expressing the at least one syntheticBCL11A microRNA described herein is expanded in vitro or ex vivo aftercryopreservation prior to administering into the subject.

In one aspect of any method, the at least one synthetic BCL11A microRNAis operably linked to a promoter and constructed in a vector forexpression in a eukaryotic cell.

In one aspect of any method, the at least one synthetic BCL11A microRNAis expressed from a RNA II polymerase.

In one aspect of any method, the at least one synthetic BCL11A microRNAis not expressed from a RNA III polymerase.

In one aspect of any method, the promoter is selected from a groupconsisting of a spleen focus-forming virus promoter, atetracycline-inducible promoter, or a β-globin locus control region anda β-globin promoter, or a hematopoietic specific promoter.

In one aspect of any method, the vector is a virus.

In one aspect of any method, the virus is a lentivirus.

In one aspect of any method, the lentivirus is selected from the groupconsisting of: human immunodeficiency virus type 1 (HIV-1), humanimmunodeficiency virus type 2 (HIV-2), caprine arthritis-encephalitisvirus (CAEV), equine infectious anemia virus (EIAV), felineimmunodeficiency virus (FIV), bovine immune deficiency virus (BIV), andsimian immunodeficiency virus (SIV).

In one aspect of any method, the subject is an animal, human ornon-human, and rodent or non-rodent. For example, the subject can be anymammal, e.g., a human, other primate, pig, rodent such as mouse or rat,rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or anon-mammal such as a bird.

In one aspect of any method, the method comprises obtaining a sample ora population of embryonic stem cells, somatic stem cells, progenitorcells, bone marrow cells, hematopoietic stem cells, or hematopoieticprogenitor cells from the subject.

In one embodiment, the embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells,hematopoietic progenitor cells are isolated from the host subject,transfected, cultured (optional), and transplanted back into the samehost, i. e. an autologous cell transplant. In another embodiment, theembryonic stem cells, somatic stem cells, progenitor cells, bone marrowcells, hematopoietic stem cells, or hematopoietic progenitor cells areisolated from a donor who is an HLA-type match with a host (recipient)who is diagnosed with or at risk of developing a hemoglobinopathy.Donor-recipient antigen type-matching is well known in the art. TheHLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent theminimum number of cell surface antigen matching required fortransplantation. That is the transfected cells are transplanted into adifferent host, i.e., allogeneic to the recipient host subject. Thedonor's or subject's embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells can be transfected with a vector ornucleic acid comprising the nucleic acid molecule described herein, thetransfected cells are culture expanded, and then transplanted into thehost subject. In one embodiment, the transplanted cells engrafts in thehost subject. The transfected cells can also be cryopreserved aftertransfected and stored, or cryopreserved after cell expansion andstored.

As used herein, treating or reducing a risk of developing ahemoglobinopathy in a subject means to ameliorate at least one symptomof hemoglobinopathy. In one aspect, the invention features methods oftreating, e.g., reducing severity or progression of, a hemoglobinopathyin a subject. In another aspect, the methods can also be used to reducea risk of developing a hemoglobinopathy in a subject, delaying the onsetof symptoms of a hemoglobinopathy in a subject, or increasing thelongevity of a subject having a hemoglobinopathy. In one aspect, themethods can include selecting a subject on the basis that they have, orare at risk of developing, a hemoglobinopathy, but do not yet have ahemoglobinopathy, or a subject with an underlying hemoglobinopathy.Selection of a subject can include detecting symptoms of ahemoglobinopathy, a blood test, genetic testing, or clinical recordings.If the results of the test(s) indicate that the subject has ahemoglobinopathy, the methods also include administering thecompositions described herein, thereby treating, or reducing the risk ofdeveloping, a hemoglobinopathy in the subject. For example, a subjectwho is diagnosis of SCD with genotype HbSS, HbS/β0 thalassemia, HbSD, orHbSO, and/or HbF<10% by electrophoresis.

As used herein, the term “hemoglobinopathy” refers to a conditioninvolving the presence of an abnormal hemoglobin molecule in the blood.Examples of hemoglobinopathies include, but are not limited to, SCD andTHAL. Also included are hemoglobinopathies in which a combination ofabnormal hemoglobins is present in the blood (e.g., sickle cell/Hb-Cdisease). An exemplary example of such a disease includes, but is notlimited to, SCD and THAL. SCD and THAL and their symptoms are well-knownin the art and are described in further detail below. Subjects can bediagnosed as having a hemoglobinopathy by a health care provider,medical caregiver, physician, nurse, family member, or acquaintance, whorecognizes, appreciates, acknowledges, determines, concludes, opines, ordecides that the subject has a hemoglobinopathy.

The term “SCD” is defined herein to include any symptomatic anemiccondition which results from sickling of red blood cells. Manifestationsof SCD include: anemia; pain; and/or organ dysfunction, such as renalfailure, retinopathy, acute-chest syndrome, ischemia, priapism, andstroke. As used herein the term “SCD” refers to a variety of clinicalproblems attendant upon SCD, especially in those subjects who arehomozygotes for the sickle cell substitution in HbS. Among theconstitutional manifestations referred to herein by use of the term ofSCD are delay of growth and development, an increased tendency todevelop serious infections, particularly due to pneumococcus, markedimpairment of splenic function, preventing effective clearance ofcirculating bacteria, with recurrent infarcts and eventual destructionof splenic tissue. Also included in the term “SCD” are acute episodes ofmusculoskeletal pain, which affect primarily the lumbar spine, abdomen,and femoral shaft, and which are similar in mechanism and in severity.In adults, such attacks commonly manifest as mild or moderate bouts ofshort duration every few weeks or months interspersed with agonizingattacks lasting 5 to 7 days that strike on average about once a year.Among events known to trigger such crises are acidosis, hypoxia, anddehydration, all of which potentiate intracellular polymerization of HbS(J. H. Jandl, Blood: Textbook of Hematology, 2nd Ed., Little, Brown andCompany, Boston, 1996, pages 544-545).

As used herein, “THAL” refers to a hereditary disorder characterized bydefective production of hemoglobin. In one embodiment, the termencompasses hereditary anemias that occur due to mutations affecting thesynthesis of hemoglobins. In other embodiments, the term includes anysymptomatic anemia resulting from thalassemic conditions such as severeor β-thalassemia, thalassemia major, thalassemia intermedia,α-thalassemias such as hemoglobin H disease. β-thalassemias are causedby a mutation in the β-globin chain, and can occur in a major or minorform. In the major form of β-thalassemia, children are normal at birth,but develop anemia during the first year of life. The mild form ofβ-thalassemia produces small red blood cells. Alpha-thalassemias arecaused by deletion of a gene or genes from the globin chain.

By the phrase “risk of developing disease” is meant the relativeprobability that a subject will develop a hemoglobinopathy in the futureas compared to a control subject or population (e.g., a healthy subjector population). For example, an individual carrying the genetic mutationassociated with SCD, an A to T mutation of the β-globin gene, andwhether the individual in heterozygous or homozygous for that mutationincreases that individual's risk.

The term “inhibitory RNA” is meant to include a nucleic acid moleculethat contains a sequence that is complementary to a target nucleic acid(e.g., a target microRNA) that mediates a decrease in the level oractivity of the target nucleic acid. Non-limiting examples of inhibitoryRNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, andantisense oligonucleotides. Methods of making inhibitory RNAs aredescribed herein. Additional methods of making inhibitory RNAs are knownin the art. In one embodiment, the BCL11A microRNA described herein isan inhibitory RNA that cause a decrease in the activity of BCL11A mRNA.

As used herein, “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion) of inhibiting or down-regulating gene expressionby mediating RNA interference. Interfering RNA includes, but is notlimited to, small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. As used herein, thephrase “post-transcriptional processing” refers to mRNA processing thatoccurs after transcription and is mediated, for example, by the enzymesDicer and/or Drosha.

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long. Each siRNA duplexis formed by a guide strand and a passenger strand. The endonucleaseArgonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Onceunwound, the guide strand is incorporated into the RNA InterferenceSpecificity Complex (RISC), while the passenger strand is released. RISCuses the guide strand to find the mRNA that has a complementary sequenceleading to the endonucleolytic cleavage of the target mRNA.

Retroviruses are RNA viruses that utilize reverse transcriptase duringtheir replication cycle. The term “retrovirus” refers to any knownretrovirus (e.g., type c retroviruses, such as Moloney murine sarcomavirus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammarytumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemiavirus (FLV), spumavirus, Friend, Murine Stem Cell Virus (MSCV) and RousSarcoma Virus (RSV)). “Retroviruses” of the invention also include humanT cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family ofretroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2,simian immunodeficiency virus (SW), feline immunodeficiency virus (FIV),equine immunodeficiency virus (EIV), and other classes of retroviruses.

The retroviral genomic RNA is converted into double-stranded DNA byreverse transcriptase. This double-stranded DNA form of the virus iscapable of being integrated into the chromosome of the infected cell;once integrated, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules which encode the structural proteins and enzymes needed toproduce new viral particles.

At each end of the provirus are structures called “long terminalrepeats” or “LTRs.” The term “long terminal repeat (LTR)” refers todomains of base pairs located at the ends of retroviral DNAs which, intheir natural sequence context, are direct repeats and contain U3, R,and U5 regions. LTRs generally provide functions fundamental to theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. The LTRcontains numerous regulatory signals including transcriptional controlelements, polyadenylation signals and sequences needed for replicationand integration of the viral genome. The viral LTR is divided into threeregions called U3, R and U5. The U3 region contains the enhancer andpromoter elements. The U5 region is the sequence between the primerbinding site and the R region and contains the polyadenylation sequence.The R (repeat) region is flanked by the U3 and U5 regions. The LTRcomposed of U3, R, and U5 regions, appears at both the both the 5′ and3′ ends of the viral genome. In one embodiment of the invention, thepromoter within the LTR, including the 5′ LTR, is replaced with aheterologous promoter. Examples of heterologous promoters that can beused include, for example, a spleen focus-forming virus (SFFV) promoter,a tetracycline-inducible (TET) promoter, a β-globin locus control regionand a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.

The term “lentivirus” refers to a group (or genus) of retroviruses thatgive rise to slowly developing disease. Viruses included within thisgroup include HIV (human immunodeficiency virus; including HIV type 1,and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes, i.e., T-cells.

The term “R region” refers to the region within retroviral LTRsbeginning at the start of the capping group (i.e., the start oftranscription) and ending immediately prior to the start of the poly Atract. The R region is also defined as being flanked by the U3 and U5regions. The R region plays an important role during reversetranscription in permitting the transfer of nascent DNA from one end ofthe genome to the other.

The term “promoter/enhancer” refers to a segment of DNA which containssequences capable of providing both promoter and enhancer functions. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous,”“exogenous,” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Methods and materials are described hereinfor use in the present invention; other, suitable methods and materialsknown in the art can also be used. The materials, methods, and examplesare illustrative only and not intended to be limiting. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of two embodiments of the disclosed synthetic BCL11Amicro RNA: BCL11A miR1 and BCL11A miR2 oligonucleotides. The stem/loopstructure is generated by the complementary sequences of the BCL11Atargeting sequences (in bold upper case nucleotide bases) in theoligonucleotides. The BCL11A targeting sequences are the BCL11Asegments. The stem/loop structure is then cloned in to a miR-223/miR-30background (micro RNA background). The entire miRNA/shRNA structure isthen cloned into a SFFV/LCR/TET cassette containing SIN lentiviralvector containing a transgene reporter (Venus).

FIG. 2 is a schematic diagram of lentiviral vector proviruses with SFFV,TET and LCR promoters.

FIG. 3 is a panel of two bar graphs showing that SFFV-LV efficientlyknocks down BCL11A and induces FT-globin expression.

FIG. 4 is a panel of two bar graphs depicting that LCR/TET-LVefficiently knocks down BCL11A and induces FT-globin expression.

FIG. 5 is a panel of photomicrographs and graphs showing that transducedCD34+ HSC differentiate ex vivo into erythrocytes and express HbF.

FIG. 6 is a panel of scatter plots depicting LCR-LV transduced CD34+HSCs from patients with SCD transplanted into NSG mice.

FIG. 7 is a panel of a photomicrograph and graphs showing the study ofpotential toxicity of BCL11A in lymphoid development.

FIGS. 8A-8E show the screening and evaluation of shRNAs targeting BCL11Ain pol III and pol II expression systems.

FIG. 8A. Schematic representation of RNA polymerase III (SEQ ID NOS99-100, respectively, in order of appearance) (pol III, U6 promoter,left side) and RNA polymerase II (SEQ ID NOS 101-102, respectively, inorder of appearance) (pol II, SFFV promoter, right side) driven shRNAand miRNA(223) embedded shRNA cassettes, respectively. Both expressioncassettes were engineered into lentivirus vectors. The various boxesrepresent the passenger strand, the guide strand, and the loop structureas indicated. The miRNA223 scaffold is represented with dotted line box.Different shRNA sequences targeting BCL11A were expressed in these twobackbones and evaluated for knockdown efficiency.

FIG. 8B. High-throughput screening of multiple shRNA sequences targetingvarious regions in BCL11A mRNA (XL/L-shared isoform sequences, XL-uniquecoding sequences and the 3′-UTR of XL isoform, as indicated) forknockdown efficiency using pol III-based lentivirus vectors. Bothinduction of ε-γ by qPCR and induction of mCherry reporter by FACS (as asurrogate for epsilon-y induction in a reporter cell line) were used asa functional readout for BCL11A knockdown. Normalized expression of ε-γmRNA relative to non-targeting control is plotted on y-axis and meanflorescence intensity (MFI) of mCherry expression relative toun-transduced control is plotted on x-axis. The 11 shRNAs that werefurther tested are marked with circles.

FIGS. 8C and 8D. Comparison of knockdown efficiency of selected shRNAsin pol III-based and pol II-based systems. MEL cells were transducedwith LKO vector or with LEGO vector to express the indicated shRNAs andthe transduced cells were selected either in the presence of puromycin(LKO) or sorted for Venus expression (LEGO). miR1 shRNA previouslyreported by Sankaran et al. ( ). BCL11A protein levels are shown (FIG.8C) by immunoblot with j-actin as control. XL and L show the position ofeach isoform of BCL11A protein. (FIG. 8D) Band intensity was analyzedusing ImageJ software.

FIG. 8E. Fold induction of normalized expression of ε-γ compared tonon-targeting control is measured by qPCR. Non-targeting shRNAtransduced MEL cells were used and expression set to 1. Data representmean±SD from a representative experiment of three independentexperiments conducted in triplicates. * P<0.05, **P<0.01, ***P<0.001.

FIGS. 9A-9F are data collected from small RNA sequencing analysis whichreveals differential processing between pol III vs pol II transcripts.

FIGS. 9A and 9B. Total RNA was isolated from transduced, sorted orpuromycin selected MEL cells expressing either miR1 or C4 shRNA. Theresulting RNA was then subjected to RNA deep-sequencing. Processed finalguide and passenger strand sequence transcribed from (FIG. 9A) pol III(LKO) (SEQ ID NO: 103) or (FIG. 9B) from pol II (LEGO) (SEQ ID NO: 104)are represented on the x-axis and corresponding number of reads permillion of total reads of each strand are plotted on the y-axis.

FIGS. 9C-9F. The sequences of processed variant guide strands of miR1transcribed from (FIG. 9C) pol III promoter (SEQ ID NOS 105-115,respectively, in order of appearance) or (FIG. 9D) pol II promoter (SEQID NOS 116-126, respectively, in order of appearance) are plotted on they-axis with the number of total reads plotted on the x-axis. Thesequence of processed variant guide strand species of C4 transcribedfrom (FIG. 9E) pol III promoter (SEQ ID NOS 127-134, respectively, inorder of appearance) or (FIG. 9F) pol II promoter (SEQ ID NOS 135, 129,132, and 136-140, respectively, in order of appearance) are plotted ony-axis with the number of total reads plotted on x-axis.

FIGS. 10A-10D show that modification of shRNA sequences leads toincreased knockdown and improved guide vs passenger strand ratio.

FIG. 10A. mIR1 and C4 shRNAs (SEQ ID NOS 25, 34, 33, and 42,respectively, in order of appearance) were modified such that four 5′bases were deleted and GCGC was added on 3′ end to yield modified shRNAtermed miR1 G5 and C4G5.

FIG. 10B. Comparison of knockdown efficiency of modified and parentshRNA sequences. MEL cells were transduced with LEGO to express theindicated shRNAs via pol II promoter and transduced cells were sortedfor Venus expression. BCL11A protein levels were measured by immunoblotwith j-actin as a loading control. XL and L indicate position of theseisoforms of BCL11A protein.

FIG. 10C. Immunoblot band intensity was analyzed using ImageJ software.

FIG. 10D. Fold induction of normalized expression compared tonon-targeting control of ε-γ by modified/unmodified shRNA sequencesmeasured by qPCR. Data represent mean±SD from a representativeexperiment of three independent experiments conducted in triplicateshowing similar results. *P<0.05, **P<0.01.

FIGS. 11A-11C show the RNA sequencing analysis of four base-pairsmodified shRNAs exhibit faithful processing.

FIG. 11A. Total small RNA (SEQ ID NO: 141) was isolated from transduced,sorted MEL expressing modified miR1 and modified C4 shRNAs andsequenced. Frequency distribution of processed guide strand species ofmodified miR1 (miR1-G5 and C4G5) transcribed from pol II promoter areplotted on the y-axis with the proportion of reads per million totalreads plotted on y-axis.

FIGS. 11B and 11C. The sequence of processed variant guide strandspecies of mIR1-G5 (SEQ ID NOS 142-150, respectively, in order ofappearance) and C4-G5 (SEQ ID NOS 151-156, 129, and 157, respectively,in order of appearance) are displayed on the y-axis and the frequency ofreads are shown on the x-axis.

FIG. 12A. Candidates from the shRNA screen targeting BCL11A using pLKOvector.

FIG. 12B. Guide strand sequence composition and distribution in PLKO.With pLKO constructs there is always a shift at the 5′ end which may bedue to extension of T rich sequence at the 3′ end. The added T's arepart of the pol III termination sequence. This shift in mature shRNAsequence indicates that during Dicer-mediated processing the 3′ countingrule is dominant, meaning cleavage of the shRNA is initiated 21nt fromthe 3′-end. This results in a 3 or 4 basepair shift at the 5′end andalso in an identically shifted seed-region (bases 2-7 of the guidestrand) which is for target recognition. FIG. 12B discloses the“mIR1PLKO” sequences as SEQ ID NOS 158 and 117-126, the “D8 PLKO”sequences as SEQ ID NOS 159-169, the “E3 PLKO” sequences as SEQ ID NOS170-180, the “B11 PLKO” sequences as SEQ ID NOS 181-191, the “C4 PLKO”sequences as SEQ ID NOS 192, 129, 132, 136-140, 128, 131, and 193, the“50A5 PLKO” sequences as SEQ ID NOS 194, 137, 195, 128, 196, 137,197-198, 195, 128, and 199, and the “B5 PLKO” sequences as SEQ ID NOS200-210, all respectively, in order of appearance.

FIG. 13. Guide strand sequence composition and distribution in LEGO.Small RNA deep sequencing analysis reveals differential processingbetween pol III vs pol II transcripts. With lego constructs there is noshift at the 5′ end and the guide strand is faithfully processed bydicer which results in the predicted product. Accordingly, the finalguide strand differs between pol III and pol II driven constructs. FIG.13 discloses the “mIR1LEGO” sequences as SEQ ID NOS 105-115, the “D8LEGO” sequences as SEQ ID NOS 211-221, the “E3 LEGO” sequences as SEQ IDNOS 222-232, the “B11 LEGO” sequences as SEQ ID NOS 233-243, the “C4LEGO” sequences as SEQ ID NOS 127-134 and 244-246, the “50A5 LEGO”sequences as SEQ ID NOS 247, 134, and 248-256, and the “B5 LEGO”sequences as SEQ ID NOS 257-267, all respectively, in order ofappearance.

FIG. 14A. Design of new shRNAs to mimic mature guide strands produced inpLKO vector. All shRNAs were modified such that four bases on the 5′were deleted and GCGC was added on 3′ end to yield modified shRNA termedmiR1G5, E3G5, B5G5, D8G5, B11G5, 50D12G5, 50B11G5, 50A5G5, 50C4G5. Withincorporation of this shift, significant improvement was observed withE3G5, D8G5 and C4G5 regarding the BCL11A knockdown and epsilon-gammainduction. The “xxxx” represents the position of the 4-base pair (bp)frame shift that results in the 4-bases removed from the unmodifiedmiR1, E3, B5, D8, B11, 50D12 (also referred to as D12), 50B11, 50A5(also referred to as A5), and 50C4 (also referred to as C4).

FIG. 14B. Guide strand sequence composition and distribution in modifiedLEGO. RNA deep sequencing analysis of modified shRNAs shows faithfulprocessing with a 4 bp shift, which indicates that by introducing theshift we are able to perfectly mimic the product of pLKO-vectors. AspLKO vectors were used for screening of effective shRNAs, thismodification mimics the precise mature product guide sequence whentransferring the shRNA cassette into pol II driven backbones. FIG. 14Bdiscloses the “mIR1MODIFIED” sequences as SEQ ID NOS 142-144, 144-150,and 268, the “D8 MODIFIED” sequences as SEQ ID NOS 269-279, the “E3MODIFIED” sequences as SEQ ID NOS 280, 177, 281-282, 175, and 283-285,the “B11 MODIFIED” sequences as SEQ ID NOS 286-296, the “C4 MODIFIED”sequences as SEQ ID NOS 297, 152-156, 129, 157, 298-299, and 132, the“50A5 MODIFIED” sequences as SEQ ID NOS 300-310, and the “B5 MODIFIED”sequences as SEQ ID NOS 311-321, all respectively, in order ofappearance.

FIG. 15. Comparison of BCL11A knockdown with modified Guide sequences.Comparison of knockdown efficiency of modified and parent shRNAsequences. Western blot showing BCL11A expression (XL and L-isoforms,top panel). Red circles indicate shRNAs where an improved BCL11Aknockdown was achieved upon introduction of a 4 bp shift. Bottom panel:Fold induction of normalized expression of ε-γ by modified/unmodifiedShRNA sequences were compared to nontargeting control as measured byqPCR.

FIG. 16. Comparison of miR expression with modified Guide sequences.Consistent with the increase in knockdown efficiency and epsilon-yinduction, the guide strand expression was high (which leads to increasein knockdown efficiency) when northern was performed in modifiedconstructs compared to unmodified especially with E3G5, D8G5 and C4G5.

FIG. 17. BCL11A knockdown efficiency and εγ induction with LEGO vectors.Comparison of knockdown efficiency of selected shRNAs in pLKO polIII-based and pLEGO pol II-based systems. MEL cells were transduced withindicated shRNAs either in pLKO vector or with pLEGO vector and thetransduced cells were selected either in the presence of puromycin(pLKO) or sorted for Venus expression (pLEGO). BCL11A protein levelswere measured by immunoblot with j-actin as control. Fold induction ofnormalized ε-γ compared to non-targeting control is measured by qPCR.Non-targeting shRNA transduced MEL cells were used as negative controls.Frame shift has strong effect on both knockdown efficiency and ε-γinduction. shRNAs targeting XL isoform alone have strong effect on εγinduction. Data represent mean±SD from three independent experiments,each conducted in triplicates. * P<0.05.

FIG. 18 shows the differential processing in pol-III shRNA vectors andpol-II microRNA adapted shRNA vectors. FIG. 18 discloses the “miR1”sequences as SEQ ID NOS 107, 117, 106, and 143, the “E3” sequences asSEQ ID NOS 223, 171, 223, and 177, the “D8” sequences as SEQ ID NOS 214,160, 212, and 270, the “B11” sequences as SEQ ID NOS 234, 182, 234, and287, the “B5” sequences as SEQ ID NOS 259, 201, 258, and 312, the “XLC4”sequences as SEQ ID NOS 131, 129, 128, and 152, and the “XLA5” sequencesas SEQ ID NOS 255, 137, 134, and 301, all respectively, in order ofappearance.

FIGS. 19A-19D show the screening and evaluation of shRNAs targetingBCL11A in pol III and pol II expression systems.

FIG. 19A. Schematic representation of LKO-U6-BCL11A-shRNA (left side)and LEGO-SFFV-BCL11A-shRNAmiR (right side). Both expression cassetteswere engineered into lentiviral vectors as described in Material andMethods. The light grey boxes represent the sense strand; white boxesrepresent the antisense strand; dark grey boxes represent the loopstructure and the miRNA223 scaffold is indicated by a dotted line. Thehairpin structures are shown below (SEQ ID NOS 322-323, respectively, inorder of appearance). Different shRNA sequences targeting BCL11A wereexpressed in these two backbones and evaluated for knockdown efficiency.

FIG. 19B. High-throughput screening of multiple shRNA sequencestargeting BCL11A mRNA for knockdown efficiency using pol III-basedlentivirus vectors. Both induction of Hbb-y mRNA by qRT-PCR andinduction of mCherry reporter by FACS (as a surrogate for ε-γ inductionin a reporter cell line) were used as a functional readout for BCL11Aknockdown. Normalized expression of Hbb-y mRNA relative to non-targetingcontrol is plotted on y-axis and fold induction of mCherry expression(by mean fluorescence intensity, MFI) relative to non-transduced controlis plotted on x-axis. The eight best performing shRNAs isolated from thescreen were further tested and are labeled as 1 through 8.

FIG. 19C. Comparison of knockdown efficiency of selected shRNAs in polIII (U6)- and pol II (SFFV) -based systems. MEL cells were transducedwith U6- (top panel) or with SFFV-(bottom panel) vectors to express theindicated shRNAs and the transduced cells were selected either in thepresence of puromycin (pol III) or sorted for Venus expression (pol II).BCL11A protein levels are shown by immunoblot with j-actin as control.XL and L on left of panel denote the position of each isoform of BCL11Aprotein.

FIG. 19D. Fold induction of normalized expression of Hbb-y compared tonon-targeting control measured by qPCR. Expression in non-targeting (NT)shRNA transduced MEL cells was set 1. Black bars represent the relativeexpression by U6 promoter driven shRNAs and white bars represent SFFVpromoter driven shRNAs. Data represent mean±SD from a representativeexperiment of three independent experiments conducted in triplicates. *P<0.05.

FIGS. 20A and 20B shows the small RNA sequencing analysis revealsdifferential processing between pol III versus pol II transcripts. SmallRNA sequencing results of MEL cells transduced with U6-shRNAs andSFFV-shRNAmiRs1, 2, 3, 4, 7, or 8. The RNA sequences were aligned to thecorresponding reference guide strand sequence, shown at the top of eachpanel in bold and the flanking sequences in grey. Different variants ofguide strands produced from (FIG. 20A) U6-shRNAs or (FIG. 20B)SFFV-shRNAmiRs are plotted on the y-axis. The relative % contribution ofeach variant is indicated on the x-axis calculated based on the totalnumber of reads matching the reference shRNA sequence. FIG. 20Adiscloses the “shRNA1” sequences as SEQ ID NOS 170-180, the “shRNA2”sequences as SEQ ID NOS 200-210, the “shRNA3” sequences as SEQ ID NOS159-169, the “shRNA4” sequences as SEQ ID NOS 181-191, the “shRNA6”sequences as SEQ ID NOS 194, 137, 195, 128, 196, 137, 197-198, 195, 128,and 199, and the “shRNA8” sequences as SEQ ID NOS 192, 129, 132,136-140, 128, 131, and 193, all respectively, in order of appearance.FIG. 20B discloses the “shRNA1” sequences as SEQ ID NOS 222-232, the“shRNA2” sequences as SEQ ID NOS 257-267, the “shRNA3” sequences as SEQID NOS 324 and 212-221, the “shRNA4” sequences as SEQ ID NOS 233-238,325, 234, and 241-243, the “shRNA6” sequences as SEQ ID NOS 247, 134,and 248-256, and the “shRNA8” sequences as SEQ ID NOS 127-134 and244-246, all respectively, in order of appearance.

FIGS. 21A-21E show the modification of shRNA sequences leads toincreased knockdown and improved guide vs. passenger strand ratio in MELcells.

FIG. 21A. SFFV-shRNAmiRs were modified by deleting the first four basesfrom the guide sequence and the addition of GCGC to the 3′ end (shRNAmodified). FIG. 21A discloses SEQ ID NOS 26, 35, 27, 36, 28, 37, 29, 38,30, 39, 31, 40, 32, 41, 33, and 42, respectively, in order ofappearance.

FIG. 21B. Comparison of knockdown efficiency of modified and parentalshRNAmiR sequences expressed from a SFFV-pol II promoter in MEL cells.BCL11A protein levels were measured in FACS sorted transduced cells byimmunoblot with β-actin as a loading control. XL and L on the left oftop panel indicate the position of these isoforms of BCL11A protein.PIII: pol III promoter vector; PII: pol II promoter vector; PIIM: pol IIpromoter vector containing modified shRNAmiR sequences.

FIG. 21C. Fold induction of Hbb-y compared to the non-targeting controlby unmodified (white bars) and modified (shaded bars) shRNAmiR sequencesmeasured by qRT-PCR. Data represent mean±SD. **P<0.01.

FIG. 21D. Northern blot analysis of total RNA extracted from cellstransduced with multiple shRNAs and shRNAmiRs. Probes (20nt)complementarity to the guide and passenger strands from positions 1 to20 of shRNAs and shRNAmiRs were utilized to measure the abundance ofprocessed small RNAs. A probe complementary to 5S RNA was used as aninternal control to determine RNA loading. PIII: pol III promotervector; PII: pol II promoter vector; PIIM: pol II promoter vectorcontaining modified shRNAmiR sequences.

FIG. 21E. RNA-sequencing results of homogeneous populations oftransduced MEL cells expressing shRNA1, 2, 3, 4, 7, or 8. The sequencesof these RNAs were aligned to the corresponding reference guide strandsequence shown at the top of each panel. The sequences of differentguide strand species are displayed on the y-axis and the frequency aspercentage of aligned reads are shown on the x-axis. FIG. 21E disclosesthe “shRNA #1” sequences as SEQ ID NOS 280, 177, 281-282, 175, and283-285, the “shRNA #2” sequences as SEQ ID NOS 311-321, the “shRNA #3”sequences as SEQ ID NOS 269-279, the “shRNA #4” sequences as SEQ ID NOS286-296, the “shRNA #6” sequences as SEQ ID NOS 300-310, and the “shRNA#8” sequences as SEQ ID NOS 297, 152-156, 129, 157, 298-299, and 132,all respectively, in order of appearance.

FIGS. 22A-22E show the modified shRNAmiRs lead to increased BCL11Aknockdown efficiency and gamma globin induction in human CD34+ derivederythroid cells.

FIG. 22A. CD34+ cells transduced with pol III or pol II vectorsexpressing different shRNAs with and without modification were selectedeither in the presence of puromycin (pol III) or sorted for Venusexpression (pol II and pol II modified). BCL11A expression was measuredby immunoblot with 3-actin as a loading control on day 11 ofdifferentiation.

FIG. 22B. Induction of γ-globin mRNA was determined on day 18 ofdifferentiation by qRT-PCR. Data represents the percentage of γ-globinof total β-locus output (γ+β-globin) for pol III (black bars), pol II(white bars), and modified pol II (grey bars). * p<0.05; *** p<0.001.

FIG. 22C. Quantification and statistical analysis of erythroiddifferentiation markers (CD71, GpA) and enucleation were assessed byflow cytometry. CTRL: control vectors SFFV-shRNAmiRNT and SEW; PIII: polIII vectors; PII: pol II vectors; PIIM: pol II vectors containingmodified shRNAmiR sequences. Data represents mean±SD from threeindependent experiments. *** p<0.001.

FIG. 22D. Hemoglobin F of cell lysates was measured by HPLC on day 18 ofdifferentiation. The arrow indicates the HbF peaks and the percentage ofHbF of total hemoglobin is shown below the chromatogram.

FIG. 22E. Correlation graph of γ-globin mRNA expression assessed byqRT-PCR versus HbF by HPLC. Black circles represent pol III vectors,open and grey circles represent pol II or modified shRNAmiRs,respectively. Correlation coefficient (r2) is shown for all data.

FIGS. 23A-23I show the negative impact of BCL11A knockdown on HSCs invivo is prevented by restricting expression to erythroid cells.

FIG. 23A. Lineage negative bone marrow cells isolated from β-YAC mice(CD45.2) were transduced ex vivo with LeGO vectors expressing shRNAmiR*targeting BCL11A or a non-targeting control vector (SFF-shRNAmiRNT) andtransplanted into lethally irradiated BoyJ recipients (CD45.1).Untransduced control cells were transplanted as control. Engraftmentanalysis was performed 4, 8 and 12 weeks post transplantation inperipheral blood and bone marrow, respectively. (n=4 mice per group)

FIG. 23B. The fraction of gene modified cells (Venus+ cells) in thesemice was determined 4, 8 and 12 weeks post transplantation in peripheralblood and bone marrow.

FIG. 23C. Competitive transplants were performed using CD45.1 and CD45.2donor cells transduced with the indicated vectors and transplanted intoCD45.1/2 heterozygous mice (top panels). Alternatively, a neutral vectorencoding blue fluorescent protein (SFFV-BFP) was used to identify thecompetitor population in a CD45.1 donor into CD45.2 recipient setting(lower panels). Shown are representative dot blots of different mixedpopulations used for transplantation three days post transduction. Thetwo competing vectors are indicated above each panel, the first oneindicates the CD45.2 or SFFV-BFP transduced populations, respectively.

FIG. 23D. The contribution of gene modified cells in competitivelyrepopulated mice was analyzed at 4 and 8 weeks post transplantation inperipheral blood (PB) or at week 12 in bone marrow (BM) and spleen(Spl). The relative contribution of gene modified cells transduced withthe two competing vectors is shown. The first vector mentioned dominatedthe hematopoietic output. Each dot represents an individual recipientmouse.

FIG. 23E. A pairwise comparison of the bone marrow B-cell fractionwithin the transduced fraction of cells between BCL11A targeting vectorsversus control vectors (SFF-shRNAmiRNT and SFFV-BFP, left panel).Similarly, the LSK content within transduced cell fractions wasanalyzed. Each dot represents an individual recipient. * and ** indicatep-values <0.05 and 0.01, respectively.

FIG. 23F. Configuration of the LCR-shRNAmiR vector used for erythroidspecific expression (details in text).

FIG. 23G. The in vivo expression profile of the LCR-vector was analyzedin various hematopoietic lineages 12 weeks after transplantation. Thepercentages of Venus+ cells in each mouse were normalized toCD71+/Ter119+ erythroid cells (n=4).

FIG. 23H. A competitive transplantation experiment as described in c andd was performed using the LCR or SFFV vectors expressing shRNAmiR*. Eachdot represents an individual recipient.

FIG. 23I. Mobilized peripheral blood CD34+ cells were transduced withLCR-shRNAmiR*, 3 and 8 or a SFFV-GFP mock vector and subjected toerythroid differentiation in vitro. At day 7 after transduction thepromoter activity of SFFV-GFP and LCR-vectors in different erythroidsubpopulation was assessed. Representative flow diagrams are shown.Error bars in all figures=SD. Statistical analysis: t-test.

FIGS. 24A-24F show the lineage specific BCL11A knockdown and gammaglobin induction by modified shRNAmiRs.

FIG. 24A. CD34+ HSPCs transduced with LCR-shRNAmiR 3, 8 or the SFFV-GFPmock vector were FACS-sorted for fluorescent reporter expression andBCL11A expression was measured by immunoblot with β-actin as a loadingcontrol on day 11 of differentiation.

FIG. 24B. Induction of γ-globin mRNA was determined on day 18 ofdifferentiation by qRT-PCR. Data represents the percentage of γ/(γ+β)globin.

FIG. 24C. Quantification and statistical analysis of erythroiddifferentiation markers (CD71, GpA) and enucleation by flow cytometricanalysis. CTRL: SFFV-GFP control vector; LCRM: Modified shRNAmiRs shownin FIG. 23A expressed via LCR promoter. Data represents mean±SD fromthree independent experiments.

FIG. 24D. HbF level of cell lysates was measured by HPLC on day 18 ofdifferentiation. Arrows indicate the HbF peaks and the percentage of HbFof total hemoglobin is shown below the chromatogram.

FIG. 24E. Correlation graph of γ-globin induction by qRT-PCR versus HbFby HPLC. Error bars indicate ±SD from three independent experiments.

FIG. 24F. Bone marrow CD34+ HSPCs were transduced with LCR-shRNAmiR3 orNT and transplanted into sublethally irradiated NSG-mice (n=3 pergroup). Untransduced cells were used as a control. Fourteen weeks laterCD34 cells were isolated from the bone marrow of transplanted animalsand subjected to erythroid differentiation in vitro for 14 days.Expression of γ-globin and β-globin was assessed in cells sorted forVenus reporter expression.

FIG. 25 shows the deep sequencing of 247 processed TRC shRNA products infour cell lines.

FIG. 26 shows the in vivo expression profile of the LCR-shRNA^(miR)vector.

FIG. 27A is a Western blot of in vitro differentiated erythroid cellsderived from transduced CD34+ cells from healthy donors showing BCL11Aisoforms (L and XL) and β-ACTIN as loading control and demonstratingeffective knock-down of BCL11A XL. VCN determined by DNA PCR is showbelow each lane.

FIG. 27B shows quantification of BCL11A knock down in erythroid cells.Data is derived from Western blots as shown in FIG. 27A. Data summarizesthree independent experiments using cells from a single donor. (Errorbars: SD)

FIG. 27C shows induction of gamma globin in erythroid cells as assessedby RT-qPCR and hemoglobin (HbF) assessed by HPLC. (Error bars: SD)

FIG. 28 shows induction of gamma globin in erythroid cells as assessedby RT-qPCR. The amount of gamma globin induction in the erythroid cellsis a measure of the in vivo BCL11A knockdown in the cells. Error bars:SD. Data from three transplanted animals per group is shown.

FIG. 29A shows Western blots showing of BCL11A (L and XL isoforms) and0-ACTIN as loading control and demonstrates effective knock-down ofBLC11A-XL. Each panel (labeled 1-6 below the lane) represents anindependent experiment using cells from a single donor.

FIG. 29B shows quantification of BCL11A knock down in erythroid cells.Data is derived from Western blots shown in FIG. 29A. (Error bar: SD)

FIG. 29C shows resulting induction of HbF by HPLC. (Error bars: SD)

FIG. 30 shows the sequences used in both SFFV and LCR backbones for theknockdown of BCL11A in CD34+ differentiated erythroid cells.

FIG. 31 shows Western blots of the BCL11A knockdown in CD34+differentiated erythroid cells.

DETAILED DESCRIPTION

The disclosure described herein is based, in part, on development oflentiviral gene therapy vectors that selectively express theBCL11A-targeting shRNA in progeny of hematopoietic stem cells (HSC).Accordingly, the disclosure encompasses novel methods for the regulationof γ-globin expression in erythroid cells. More specifically, theseactivities can be harnessed in methods for the treatment ofhemoglobinopathies, including SCD and THAL, by induction of γ-globin viainhibition of the BCL11A gene product. In particular embodiments,lentiviral gene therapy vectors that selectively express theBCL11A-targeting shRNA in progeny of HSCs, hematopoietic progenitorcells, or other stem cells such as embryonic cells are provided.

Normal adult hemoglobin comprises four globin proteins, two of which arealpha (a) proteins and two of which are beta (β) proteins. Duringmammalian fetal development, particularly in humans, the fetus producesfetal hemoglobin, which comprises two gamma (γ)-globin proteins insteadof the two β-globin proteins. At some point during fetal development, aglobin fetal switch occurs at which point erythrocytes in the fetusswitch from making predominantly γ-globin to making predominantlyβ-globin. The developmental switch from production of predominantlyfetal hemoglobin or HbF (α2γ2) to production of adult hemoglobin or HbA(α2β2) begins at about 28 to 34 weeks of gestation and continues shortlyafter birth until HbA becomes predominant. This switch results primarilyfrom decreased transcription of the γ-globin genes and increasedtranscription of β-globin genes. On average, the blood of a normal adultcontains only about 2% HbF, though residual HbF levels have a varianceof over 20-fold in healthy adults (Atweh, Semin. Hematol. 38:367-73(2001)).

Hemoglobinopathies encompass a number of anemias of genetic origin inwhich there is a decreased production and/or increased destruction(hemolysis) of red blood cells (RBCs). These also include geneticdefects that result in the production of abnormal hemoglobins with aconcomitant impaired ability to maintain oxygen concentration. Some suchdisorders involve the failure to produce normal β-globin in sufficientamounts, while others involve the failure to produce normal β-globinentirely. These disorders associated with the β-globin protein arereferred to generally as hemoglobinopathies. For example, SCD resultsfrom a point mutation in the β-globin structural gene, leading to theproduction of abnormal (sickled) hemoglobin (HbS). HbS RBCs are morefragile than normal RBCs and undergo hemolysis more readily, leadingeventually to anemia (Atweh, Semin. Hematol. 38(4):367-73 (2001)). THALresults from a partial or complete defect in the expression of theβ-globin gene, leading to deficient or absent HbA.

The search for treatment aimed at reduction of globin chain imbalance inpatients with hemoglobinopathies has focused on the pharmacologicmanipulation of fetal HbF. The therapeutic potential of such approachesis demonstrated by observations that certain populations of adultpatients with R chain abnormalities and higher than normal levels of HbFexperience a milder clinical course of disease than patients with normaladult levels of HbF. For example, a group of Saudi Arabian sickle cellanemia patients who express 20-30% HbF have only mild clinicalmanifestations of the disease (Pembrey, et al., Br. J. Haematol. 40:415-429 (1978)). It is now accepted that hemoglobinopathies, such as SCDand THAL, are ameliorated by increased HbF production (Jane et al., Br.J. Haematol. 102: 415-422 (1998); Bunn, N. Engl. J. Med. 328: 129-131(1993)).

The transcriptional repressor BCL11A represents a therapeutic target for(3-hemoglobinopathies. RNA interference was applied using pol IIIpromoter-expressed short hairpin RNAs (shRNAs) to reduce BCL11Aexpression in hematopoietic cells. Knockdown of BCL11A in murinehematopoietic stem cells (HSCs) impaired long-term engraftment. To avoidHSC toxicity, the expression of BCL11A in erythroid cells wasselectively suppressed via pol II promoter expressed microRNA adaptedshRNAs (shRNAmiRs). With identical target matched sequences, markedlyreduced knockdown was observed using pol II vectors due to 3-5 ntdifferences in the guide strands between the systems that stronglyinfluence target knockdown. A corresponding 4 nt shift was engineeredinto guide strands of shRNAmiRs that surprisingly and unexpectedlyimproved the knockdown of BCL11A and derepression of Hbb-y, a functionalhomolog of the human γ-globin gene in a murine erythroid cell line. Themodified shRNAmiRs were expressed in an erythroid-specific fashion tocircumvented the adverse effects on murine HSC engraftment, and this ledto efficient BCL11A knockdown and high levels of HbF in humanCD34-derived erythroid cells. A strategy was developed for theprospective design of shRNAmiRs derived from pol III-expressed shRNAscreens. This strategy constitutes an improved approach to genetictherapy in hemoglobinopathies and other diseases requiringlineage-specific expression of gene silencing sequences.

Retroviral and Lentiviral Vectors

In some embodiments, the present disclosure provides improvedcompositions and methods for treating hemoglobinopathies usingretrovirus-based, e.g., lentivirus-based, gene delivery vectors thatachieve sustained, high-level expression of transferred therapeuticgenes in eythroid cells or erythroid precursor cells. In one embodimentof the invention, the vector comprises an artificial miRNA comprisingtargeting sequences to BCL11A cloned into the stem loop of theendogenouse miR-223 sequence (Amendola et al., Mol Ther 17:1039-52,2009). The stem/loop structure of the present vectors are generated bycomplementary sequences of the oligonucleotides of SEQ ID NOs:1-18 and25-44 disclosed herein. See FIGS. 1, 12A, 14A, 21A, and EXAMPLE 11. Thisstem/loop structure was cloned into a miR-223/miR-30 background. Theentire miRNA/shRNA structure was then cloned into a cassette with aSFFV, TET, or LCR promoter containing self-inactivating (SIN) vector.Particular lentiviral vectors of the invention are described by Pawliuket al. (2001) Science 294:2368 and Imren et al. (2002) PNAS 99:14380,incorporated by reference herein.

Accordingly, in one aspect, provided herein is a synthetic BCL11AmicroRNA comprising a first BCL11A segment, a loop segment, and a secondBCL11A segment arranged in tandem in a 5′ to 3′ direction, wherein theloop segment is between and directly linked to the first and secondBCL11A segments, and wherein the second BCL11A segment is complementaryto the first BCL11A segment such that the first and second BCL11Asegments base pair to form a hairpin loop with the loop segment formingthe loop portion of the hairpin loop thus formed.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first and second BCL11A segments are about 18 to 25nucleotides long. The first BCL11A segment is derived from a BCL11Asequence and gives rise to the passenger strand during shRNA processingto a duplex siRNA and the second BCL11A segment is complementary tofirst BCL11A segment, wherein the second BCL11A segment gives rise tothe guide strand that is incorporated into the RNA InterferenceSpecificity Complex (RISC) for RNA interference or BCL11A genesilencing.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first and second BCL11A segments are derived from BCL11AmRNA sequence.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment starts with a -GCGC- at the 5′ end andthe second BCL11A segment ends with a -GCGC- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment further consist a -GCGC- at the 5′ endand the second BCL11A segment ends with a -GCGC- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment starts with a -GCGA-, -TCTG-, or -TG-at the 5′ end and the second BCL11A segment is complementary to firstBCL11A segment.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment further consist a -GCGA-, -TCTG-, or-TG- at the 5′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the second BCL11A segment ends with a -TTTT- at the 3′ end.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA comprise a nucleotide sequenceselected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA consists of a nucleotide sequenceselected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the synthetic BCL11A microRNA consist essentially of anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the first BCL11A segment is selected from the group consistingof CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; derived from BCL11A miR1oligo described herein), CCAGAGGATGACGATTGTTTA (SEQ. ID. NO: 47; derivedfrom BCL11A miR2 oligo described herein), TCGGAGACTCCAGACAATCGC (SEQ.ID. NO: 48; derived from BCL11A E3 oligo or shRNA1 or E3 describedherein), CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 orB5 described herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50; derivedfrom shRNA4 or B11 described herein), TTCTCTTGCAACACGCACAGA (SEQ. ID.NO: 51; derived from BCL11A D8 oligo or shRNA3 or D8 described herein),GATCGAGTGTTGAATAATGAT (SEQ. ID. NO: 52; derived from shRNA5 or 50D12 o1D12 described herein), CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53; derivedfrom shRNA5 or 50A5 described herein), CACTGTCCACAGGAGAAGCCA (SEQ. ID.NO: 54; derived from shRNA7 or 50B11 described herein),ACAGTACCCTGGAGAAACACA (SEQ. ID. NO: 55; derived from BCL11A XLC4, shRNA8and 50C4 described herein), CAACAAGATGAAGAGCACCAA (SEQ. ID. NO: 56;derived from BCL11A Non-targeting oligos described herein),gcgcCGCACAGAACACTCATG (SEQ. ID. NO: 57; derived from miR1G5 oligodescribed herein), GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58; derived fromE3G5 or E3 mod oligo or shRNA1 mod described herein),gcgcCCTCCAGGCAGCTCAAA (SEQ. ID. NO: 59; derived from B5G5 or shRNA2 moddescribed herein); gcgcTCAGGACTAGGTGCAGA (SEQ. ID. NO: 60; derived fromB11G5 or shRNA4 mod described herein); gcgcGATCGAGTGTTGAATAA (SEQ. ID.NO: 61; derived from 50D12G5, D12G4 or shRNA5 mod described herein);gcgcCAGTACCCTGGAGAAAC (SEQ. ID. NO: 62; derived from 50A5G5 or shRNA6mod described herein); gcgcCACTGTCCACAGGAGAA (SEQ. ID. NO: 63; derivedfrom 50B11G5 or shRNA7 mod described herein); GCGCTTCTCTTGCAACACGCA(SEQ. ID. NO: 64; derived from BCL11A D8G5 or D8 mod or shRNA3 moddescribed herein), GCGCACAGTACCCTGGAGAAA (SEQ. ID. NO: 65; derived fromBCL11A C4G5, or C4 mod or shRNA8 mod described herein),CGCACAGAACACTCATGGATT (SEQ. ID. NO: 66; derived from BCL11A D12G5-2described herein), and ACGCTCGCACAGAACACTCATGGATT (SEQ. ID. NO: 67;derived from BCL11A D12G5-2 described herein).

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the loop segment is derived from a microRNA. In one embodiment,the microRNA is a hematopoietic specific microRNA. For examples,miR-142, miR-155, miR-181 and miR-223.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the microRNA is miR223.

In one embodiment of any one of the synthetic BCL11A microRNA describedherein, the loop segment is ctccatgtggtagag (SEQ ID NO:68).

In one aspect, the present specification provides an isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:1-18, 25-44, or a synthetic BCL11A microRNAdescribed herein.

Accordingly, in one aspect, the present specification provides acomposition comprising at least one nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ IDNOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.

Accordingly, in one aspect, the present specification provides acomposition comprising at least a vector or a bacterium comprising anucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11AmicroRNA described herein.

In one aspect, the present specification provides a host cell comprisinga vector or virus which comprises at least one nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA describedherein.

In one aspect, the present specification provides a host cell comprisinga vector, virus or a bacterium which comprises at least one nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11AmicroRNA described herein.

In one embodiment, the vector is a viral vector or a virus.

RNA interference (RNAi) mediated by short interfering RNAs (siRNA) ormicroRNAs (miRNA) is a powerful method for post-transcriptionalregulation of gene expression. RNAi has been extensively used for thestudy of biological processes in mammalian cells and could constitute atherapeutic approach to human diseases in which selective modulation ofgene expression would be desirable. Depending on the degree ofcomplementarity between miRNA and target mRNA sequences, loss of geneexpression occurs by inducing degradation of the cognate mRNA or bytranslational attenuation. Endogenous miRNAs are transcribed as primarytranscripts and subsequently processed by the RNAse III enzyme Drosha,(1) to create a stem loop structure. Nuclear export and cleavage byDicer generates a mature short double stranded molecule (siRNA) that isseparated into guide and passenger strands. The guide strand is loadedinto the RNA induced silencing complex (RISC), the effector complexmediating cleavage of target mRNAs with the functional guide strandbinding to RISC proteins (2) while the passenger strand is degraded[reviewed in (3)]. The loading of guide versus passenger strands intoRISC largely depends on the 5′ end stability of the siRNA, with the lessstable strand preferentially incorporated into RISC (4, 5), although theexact regulation in mammalian cells is incompletely understood. The 5′end of the guide strand contains the “seed region,” which is criticalfor target identification (6, 7). Precise cleavage by Drosha and Diceris critical for the generation of guide RNAs with defined seed regionsthat mediate efficient binding to the appropriate target mRNAs.Inaccurate processing results in binding to off-target molecules but ashift in cleavage sites also alters the nucleotide composition of duplexends, which may have a profound effect on strand loading into RISC (8).

The inhibiting the expression of selected target polypeptides is throughthe use of RNA interference agents. RNA interference (RNAi) uses smallinterfering RNA (siRNA) duplexes that target the messenger RNA encodingthe target polypeptide for selective degradation. siRNA-dependentpost-transcriptional silencing of gene expression involves cleaving thetarget messenger RNA molecule at a site guided by the siRNA. RNAi is anevolutionally conserved process whereby the expression or introductionof RNA of a sequence that is identical or highly similar to a targetgene results in the sequence specific degradation or specificpost-transcriptional gene silencing (PTGS) of messenger RNA (mRNA)transcribed from that targeted gene (see Coburn, G. and Cullen, B.(2002) J. Virology 76(18):9225), thereby inhibiting expression of thetarget gene. In one embodiment, the RNA is double stranded RNA (dsRNA).This process has been described in plants, invertebrates, and mammaliancells. In nature, RNAi is initiated by the dsRNA-specific endonucleaseDicer, which promotes processive cleavage of long dsRNA intodouble-stranded fragments termed siRNAs. siRNAs are incorporated into aprotein complex (termed “RNA induced silencing complex,” or “RISC”) thatrecognizes and cleaves target mRNAs. RNAi can also be initiated byintroducing nucleic acid molecules, e.g., synthetic siRNAs or RNAinterfering agents, to inhibit or silence the expression of targetgenes. As used herein, “inhibition of target gene expression” includesany decrease in expression or protein activity or level of the targetgene or protein encoded by the target gene as compared to a situationwherein no RNA interference has been induced. The decrease will be of atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or more as compared to the expression of a target gene or theactivity or level of the protein encoded by a target gene which has notbeen targeted by an RNA interfering agent.

The terms “RNA interference agent” and “RNA interference” as they areused herein are intended to encompass those forms of gene silencingmediated by double-stranded RNA, regardless of whether the RNAinterfering agent comprises an siRNA, miRNA, shRNA or otherdouble-stranded RNA molecule. siRNA is defined as an RNA agent whichfunctions to inhibit expression of a target gene, e.g., by RNAi. AnsiRNA may be chemically synthesized, may be produced by in vitrotranscription, or may be produced within a host cell. In one embodiment,siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40nucleotides in length, preferably about 15 to about 28 nucleotides, morepreferably about 19 to about 25 nucleotides in length, and morepreferably about 19, 20, 21, 22, or 23 nucleotides in length, and maycontain a 3′ and/or 5′ overhang on each strand having a length of about0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang isindependent between the two strands, i.e., the length of the overhang onone strand is not dependent on the length of the overhang on the secondstrand. Preferably the siRNA is capable of promoting RNA interferencethrough degradation or specific post-transcriptional gene silencing(PTGS) of the target messenger RNA (mRNA).

siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).In one embodiment, these shRNAs are composed of a short (e.g., about 19to about 25 nucleotide) antisense strand, followed by a nucleotide loopof about 5 to about 9 nucleotides, and the analogous sense strand.Alternatively, the sense strand may precede the nucleotide loopstructure and the antisense strand may follow. These shRNAs may becontained in plasmids, retroviruses, and lentiviruses and expressedfrom, for example, the pol III U6 promoter, or another promoter (see,e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated byreference herein in its entirety). The target gene or sequence of theRNA interfering agent may be a cellular gene or genomic sequence, e.g.,the BCL11A sequence. An siRNA may be substantially homologous to thetarget gene or genomic sequence, or a fragment thereof. As used in thiscontext, the term “homologous” is defined as being substantiallyidentical, sufficiently complementary, or similar to the target mRNA, ora fragment thereof, to effect RNA interference of the target. Inaddition to native RNA molecules, RNA suitable for inhibiting orinterfering with the expression of a target sequence include RNAderivatives and analogs. Preferably, the siRNA is identical to itstarget. The siRNA preferably targets only one sequence. Each of the RNAinterfering agents, such as siRNAs, can be screened for potentialoff-target effects by, for example, expression profiling. Such methodsare known to one skilled in the art and are described, for example, inJackson et al. Nature Biotechnology 6:635-637, 2003. In addition toexpression profiling, one may also screen the potential target sequencesfor similar sequences in the sequence databases to identify potentialsequences which may have off-target effects. For example, 15, or perhapsas few as 11 contiguous nucleotides, of sequence identity are sufficientto direct silencing of non-targeted transcripts. Therefore, one mayinitially screen the proposed siRNAs to avoid potential off-targetsilencing using the sequence identity analysis by any known sequencecomparison methods, such as BLAST. siRNA sequences are chosen tomaximize the uptake of the antisense (guide) strand of the siRNA intoRISC and thereby maximize the ability of RISC to target BCL11A mRNA fordegradation. This can be accomplished by scanning for sequences thathave the lowest free energy of binding at the 5′-terminus of theantisense strand. The lower free energy leads to an enhancement of theunwinding of the 5′-end of the antisense strand of the siRNA duplex,thereby ensuring that the antisense strand will be taken up by RISC anddirect the sequence-specific cleavage of the human BCL11A mRNA. siRNAmolecules need not be limited to those molecules containing only RNA,but, for example, further encompasses chemically modified nucleotidesand non-nucleotides, and also include molecules wherein a ribose sugarmolecule is substituted for another sugar molecule or a molecule whichperforms a similar function. Moreover, a non-natural linkage betweennucleotide residues can be used, such as a phosphorothioate linkage. TheRNA strand can be derivatized with a reactive functional group of areporter group, such as a fluorophore. Particularly useful derivativesare modified at a terminus or termini of an RNA strand, typically the 3′terminus of the sense strand. For example, the 2′-hydroxyl at the 3′terminus can be readily and selectively derivatized with a variety ofgroups. Other useful RNA derivatives incorporate nucleotides havingmodified carbohydrate moieties, such as 2′O-alkylated residues or2′-O-methyl ribosyl derivatives and 2′-O-fluoro ribosyl derivatives. TheRNA bases may also be modified. Any modified base useful for inhibitingor interfering with the expression of a target sequence may be used. Forexample, halogenated bases, such as 5-bromouracil and 5-iodouracil canbe incorporated. The bases may also be alkylated, for example,7-methylguanosine can be incorporated in place of a guanosine residue.Non-natural bases that yield successful inhibition can also beincorporated. The most preferred siRNA modifications include2′-deoxy-2′-fluorouridine or locked nucleic acid (LNA) nucleotides andRNA duplexes containing either phosphodiester or varying numbers ofphosphorothioate linkages. Such modifications are known to one skilledin the art and are described, for example, in Braasch et al.,Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications tothe siRNA molecules can be introduced using chemistries established forantisense oligonucleotide technology. Preferably, the modificationsinvolve minimal 2′-O-methyl modification, preferably excluding suchmodification. Modifications also preferably exclude modifications of thefree 5′-hydroxyl groups of the siRNA. The Examples herein providespecific examples of RNA interfering agents, such as shRNA moleculesthat effectively target BCL11A mRNA.

Polymerase (pol) III driven short hairpin RNAs (shRNAs) are mostcommonly used in biological experimental settings. shRNAs mimic thestructure of miRNA precursor intermediates, and thus bypass the firstcleavage step mediated by Drosha. shRNAs can be abundantly expressed toprovide efficient knockdown. However, at high multiplicities ofinfection (MOI), oversaturation of the endogenous RNAi machinery hasbeen reported in some cases to be associated with cytotoxic effects dueto the dysregulation of endogenous miRNAs (9-11). Two components ofmicroRNA processing, Exportin5 and Ago2, seem to limit the capacity ofthis pathway, and overexpression of these proteins results in increasedknockdown capacity (12-15). Additionally, activation of innate immuneresponses triggered by small RNAs in a sequence specific as well asnon-specific manner may mediate cytotoxic side effects (16, 17),reviewed in (18). These effects have resulted in increased mortality inmice in some experimental transgenic model systems reportedly as adirect side effect of shRNA over-expression (14, 19).

For clinical translation of RNAi based therapeutics, alternativeexpression systems utilizing polymerase II promoters will likely berequired. This class of promoters allows for utilization of appropriateregulatory elements for lineage or even cell-type specific expression.It also could provide lower levels of expression compared to pol IIIpromoters, which may obviate over-saturation of the processing machinerythat have been reported in cells transduced at high MOIs). Complicatingthe use of pol II promoters for shRNA expression, requires embedding ofthe shRNA sequences into flanking sequences usually derived fromendogenous miRNA precursors for efficient processing. shRNAs flanked bya miRNA scaffold mimic the structure of endogenous miRNAs (10, 20). Todate, flanking regions derived from human miRNA-30 and miRNA-223 havebeen widely used for incorporation of recombinant shRNAs for expressionin mammalian cells, and there have been numerous efforts to betterunderstand and to improve this expression strategy (21). The lattermiRNA has been shown to be particularly effective when used as scaffoldfor shRNA expression in hematopoietic cells and mediates substantialknockdown of target mRNAs as a result of efficient processing and lowpassenger strand activity in several hematopoietic cell types (21, 22).

In this disclosure, the inventors utilized BCL11A as a target to studythe processing and optimization of shRNAmiRs for potential therapeuticapplications. BCL11A is a validated therapeutic target for reactivationof γ-globin gene and therefore HbF expression in the majorhemoglobinopathies, sickle cell disease (SCD) and β-thalassemias. Downmodulation or genetic deletion of BCL11A relieves γ-globin repression(23) and inactivation of BCL11A in the erythroid lineage prevents SCDphenotype and organ toxicities in genetically engineered mice (24). Themouse embryonic Hbb-y gene is a functional homolog of the human γ-globingene, and therefore serves as a convenient surrogate for assessment ofthe effect of BCL11A knockdown in murine erythroleukemia (MEL) cells.Initially we observed a markedly reduced efficiency of knockdown ofBCL11A upon expression of shRNA using pol II-based as compared with polIII-based vectors. Pol III and pol II shRNAmiR designs typicallyincorporate 21 base target site matched sequences within the palindromichairpin stem, but the transcripts from these two types of expressioncassettes are expected to be processed differently (25). The pol IIshRNAmiR transcripts enter the RNAi processing pathway upstream ofDrosha processing, whereas the much shorter pol III products areexpected to enter the pathway downstream of Drosha and to be cleavedonly at the loop end by Dicer. Based upon the sequences of processedsmall RNAs derived from pol III and pol II promoters we observed thatpol III shRNA cassettes and pol II shRNAmiR cassettes yielded differentprocessed shRNAs with respect to the relative positioning of the 21 basetarget-matched sequences. Redesigned shRNAmiRs that mimicked the matureguide strand sequences produced by effective pol III-driven shRNAs ledto enhancement in processing efficiency and inhibition of the targetmRNA. Incorporation of these modifications into an erythroid-specificmammalian expression vector led to significant knockdown of BCL11Aprotein and re-induction of fetal hemoglobin. This strategy also avoidedtoxicity in the hematopoietic stem cell and B cell lineage compartmentsthat accompanied pan-hematopoietic shRNA expression. In summary, thedata demonstrate critical features of RNA processing relevant to the useof shRNA in different vector contexts, and also provide a strategy forlineage-specific gene knockdown that circumvents adverse consequences ofwidespread expression. Our findings have important implications fordesign of microRNA embedded shRNAs and their application in RNAi basedgene therapy approaches.

In one embodiment, the RNA interference agent is delivered oradministered in a pharmaceutically acceptable carrier. Additionalcarrier agents, such as liposomes, can be added to the pharmaceuticallyacceptable carrier. In another embodiment, the RNA interference agent isdelivered by a vector encoding small or short hairpin RNA (shRNA) in apharmaceutically acceptable carrier to the cells in an organ of anindividual. The shRNA is converted by the cells after transcription intosiRNA capable of targeting, for example, BCL11A.

In one embodiment, the RNA interference agent is a nucleic acid moleculecomprising the nucleotide sequence selected from the group consisting ofSEQ ID NOS: 1-18, and 25-44, or a synthetic BCL11A microRNA describedherein.

In one embodiment, the vector is a regulatable vector, such astetracycline inducible vector. Methods described, for example, in Wanget al. Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On vectors (BDBiosciences Clontech, Palo Alto, Calif.) can be used. In one embodiment,the RNA interference agents used in the methods described herein aretaken up actively by cells in vivo following intravenous injection,e.g., hydrodynamic injection, without the use of a vector, illustratingefficient in vivo delivery of the RNA interfering agents. One method todeliver the siRNAs is catheterization of the blood supply vessel of thetarget organ. Other strategies for delivery of the RNA interferenceagents, e.g., the siRNAs or shRNAs used in the methods of the invention,may also be employed, such as, for example, delivery by a vector, e.g.,a plasmid or viral vector, e.g., a lentiviral vector. Such vectors canbe used as described, for example, in Xiao-Feng Qin et al. Proc. Natl.Acad. Sci. U.S.A., 100: 183-188. Other delivery methods include deliveryof the RNA interfering agents, e.g., the siRNAs or shRNAs of theinvention, using a basic peptide by conjugating or mixing the RNAinterfering agent with a basic peptide, e.g., a fragment of a TATpeptide, mixing with cationic lipids or formulating into particles. TheRNA interference agents, e.g., the siRNAs targeting BCL11A mRNA, may bedelivered singly, or in combination with other RNA interference agents,e.g., siRNAs, such as, for example siRNAs directed to other cellulargenes. BCL11A siRNAs may also be administered in combination with otherpharmaceutical agents which are used to treat or prevent diseases ordisorders associated with oxidative stress, especially respiratorydiseases, and more especially asthma. Synthetic siRNA molecules,including shRNA molecules, can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA molecule canbe chemically synthesized or recombinantly produced using methods knownin the art, such as using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer (see, e.g.,Elbashir, S. M. et al. (2001) Nature 411:494-498; Elbashir, S. M., W.Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200; Harborth,J. et al. (2001) J. Cell Science 114:4557-4565; Masters, J. R. et al.(2001) Proc. Natl. Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al.(1999) Genes & Development 13:3191-3197). Alternatively, severalcommercial RNA synthesis suppliers are available including, but notlimited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass.,USA), and Cruachem (Glasgow, UK). As such, siRNA molecules are notoverly difficult to synthesize and are readily provided in a qualitysuitable for RNAi. In addition, dsRNAs can be expressed as stem loopstructures encoded by plasmid vectors, retroviruses and lentiviruses(Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; McManus, M. T. etal. (2002) RNA 8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol.20:505-508; Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500;Sui, G. et al. (2002) Proc. Natl. Acad. Sci., USA 99:5515-5520;Brummelkamp, T. et al. (2002) Cancer Cell 2:243; Lee, N. S., et al.(2002) Nat. Biotechnol. 20:500-505; Yu, J. Y., et al. (2002) Proc. Natl.Acad. Sci., USA 99:6047-6052; Zeng, Y., et al. (2002) Mol. Cell.9:1327-1333; Rubinson, D. A., et al. (2003) Nat. Genet. 33:401-406;Stewart, S. A., et al. (2003) RNA 9:493-501). These vectors generallyhave a polIII promoter upstream of the dsRNA and can express sense andantisense RNA strands separately and/or as a hairpin structures. Withincells, Dicer processes the short hairpin RNA (shRNA) into effectivesiRNA. The targeted region of the siRNA molecule of the presentinvention can be selected from a given target gene sequence, e.g., aBCL11A coding sequence, beginning from about 25 to 50 nucleotides, fromabout 50 to 75 nucleotides, or from about 75 to 100 nucleotidesdownstream of the start codon. Nucleotide sequences may contain 5′ or 3′UTRs and regions nearby the start codon. One method of designing a siRNAmolecule of the present invention involves identifying the 23 nucleotidesequence motif and selecting hits with at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70% or 75% G/C content. Alternatively, if no suchsequence is found, the search may be extended using the motif NA(N21),where N can be any nucleotide. In this situation, the 3′ end of thesense siRNA may be converted to TT to allow for the generation of asymmetric duplex with respect to the sequence composition of the senseand antisense 3′ overhangs. The antisense siRNA molecule may then besynthesized as the complement to nucleotide positions 1 to 21 of the 23nucleotide sequence motif. The use of symmetric 3′ TT overhangs may beadvantageous to ensure that the small interfering ribonucleoproteinparticles (siRNPs) are formed with approximately equal ratios of senseand antisense target RNA-cleaving siRNPs (Elbashir et al., (2001) supraand Elbashir et al., 2001 supra). Analysis of sequence databases,including but not limited to the NCBI, BLAST, Derwent, and GenSeq aswell as commercially available oligosynthesis companies such asOLIGOENGINE®, may also be used to select siRNA sequences against ESTlibraries to ensure that only one gene is targeted.

Lentiviral vectors of the invention include, but are not limited to,human immunodeficiency virus (e.g., HIV-1, HIV-2), felineimmunodeficiency virus (FIV), simian immunodeficiency virus (SIV),bovine immunodeficiency virus (BIV), and equine infectious anemia virus(EIAV). These vectors can be constructed and engineered usingart-recognized techniques to increase their safety for use in therapyand to include suitable expression elements and therapeutic genes, suchas those described below, which encode siRNAs for treating conditionsincluding, but not limited to, hemoglobinopathies.

In consideration of the potential toxicity of lentiviruses, the vectorscan be designed in different ways to increase their safety in genetherapy applications. For example, the vector can be made safer byseparating the necessary lentiviral genes (e.g., gag and pol) ontoseparate vectors as described, for example, in U.S. Pat. No. 6,365,150,the contents of which are incorporated by reference herein. Thus,recombinant retrovirus can be constructed such that the retroviralcoding sequence (gag, pol, env) is replaced by a gene of interestrendering the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions through the use of ahelper virus or a packaging cell line, by standard techniques. Protocolsfor producing recombinant retroviruses and for infecting cells in vitroor in vivo with such viruses can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals.

A major prerequisite for the use of viruses as gene delivery vectors isto ensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment packaging cell lines, which produce onlyreplication-defective retroviruses, has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Accordingly, in oneembodiment of the invention, packaging cell lines are used to propagatevectors (e.g., lentiviral vectors) of the invention to increase thetiter of the vector virus. The use of packaging cell lines is alsoconsidered a safe way to propagate the virus, as use of the systemreduces the likelihood that recombination will occur to generatewild-type virus. In addition, to reduce toxicity to cells that caused byexpression of packaging proteins, packaging systems can be use in whichthe plasmids encoding the packaging functions of the virus are onlytransiently transfected by, for example, chemical means.

In another embodiment, the vector can be made safer by replacing certainlentiviral sequences with non-lentiviral sequences. Thus, lentiviralvectors of the present disclosure may contain partial (e.g., split) genelentiviral sequences and/or non-lentiviral sequences (e.g., sequencesfrom other retroviruses) as long as its function (e.g., viral titer,infectivity, integration and ability to confer high levels and durationof therapeutic gene expression) are not substantially reduced. Elementswhich may be cloned into the viral vector include, but are not limitedto, promoter, packaging signal, LTR(s), polypurine tracts, and a reverseresponse element (RRE).

In one embodiment of the disclosure, the LTR region is modified byreplacing the viral LTR promoter with a heterologous promoter. In oneembodiment, the promoter of the 5′ LTR is replaced with a heterologouspromoter. Examples of heterologous promoters which can be used include,but are not limited to, a spleen focus-forming virus (SFFV) promoter, atetracycline-inducible (TET) promoter, a β-globin locus control regionand a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.

In some embodiments, the lentiviral vectors of the disclosure alsoinclude vectors which have been modified to improve upon safety in theuse of the vectors as gene delivery agents in gene therapy. In oneembodiment of the invention, an LTR region, such as the 3′ LTR, of thevector is modified in the U3 and/or U5 regions, wherein a SIN vector iscreated. Such modifications contribute to an increase in the safety ofthe vector for gene delivery purposes. In one embodiment, the SIN vectorof the invention comprises a deletion in the 3′ LTR wherein a portion ofthe U3 region is replaced with an insulator element. The insulatorprevents the enhancer/promoter sequences within the vector frominfluencing the expression of genes in the nearby genome, andvice/versa, to prevent the nearby genomic sequences from influencing theexpression of the genes within the vector. In a further embodiment ofthe invention, the 3′ LTR is modified such that the U5 region isreplaced, for example, with an ideal poly(A) sequence. It should benoted that modifications to the LTRs such as modifications to the 3′LTR, the 5′ LTR, or both 3′ and 5′ LTRs, are also included in theinvention.

The promoter of the lentiviral vector can be one which is naturally(i.e., as it occurs with a cell in vivo) or non-naturally associatedwith the 5′ flanking region of a particular gene. Promoters can bederived from eukaryotic genomes, viral genomes, or synthetic sequences.Promoters can be selected to be non-specific (active in all tissues)(e.g., SFFV), tissue specific (e.g., (LCR), regulated by naturalregulatory processes, regulated by exogenously applied drugs (e.g.,TET), or regulated by specific physiological states such as thosepromoters which are activated during an acute phase response or thosewhich are activated only in replicating cells. Non-limiting examples ofpromoters in the present invention include the spleen focus-formingvirus promoter, a tetracycline-inducible promoter, a β-globin locuscontrol region and a β-globin promoter (LCR), a cytomegalovirus (CMV)promoter, retroviral LTR promoter, cytomegalovirus immediate earlypromoter, SV40 promoter, and dihydrofolate reductase promoter. Thepromoter can also be selected from those shown to specifically expressin the select cell types which may be found associated with conditionsincluding, but not limited to, hemoglobinopathies. In one embodiment ofthe invention, the promoter is cell specific such that gene expressionis restricted to red blood cells. Erythrocyte-specific expression isachieved by using the human β-globin promoter region and locus controlregion (LCR).

Skilled practitioners will recognize that selection of the promoter toexpress the polynucleotide of interest will depend on the vector, thenucleic acid cassette, the cell type to be targeted, and the desiredbiological effect. Skilled practitioners will also recognize that in theselection of a promoter, the parameters can include: achievingsufficiently high levels of gene expression to achieve a physiologicaleffect; maintaining a critical level of gene expression; achievingtemporal regulation of gene expression; achieving cell type specificexpression; achieving pharmacological, endocrine, paracrine, orautocrine regulation of gene expression; and preventing inappropriate orundesirable levels of expression. Any given set of selectionrequirements will depend on the conditions but can be readily determinedonce the specific requirements are determined. In one embodiment of theinvention, the promoter is cell-specific such that gene expression isrestricted to red blood cells. Erythrocyte-specific expression isachieved by using the human β-globin promoter region and locus controlregion (LCR).

Standard techniques for the construction of expression vectors suitablefor use in the present invention are well-known to those of ordinaryskill in the art and can be found in such publications as Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold SpringHarbor, N.Y. A variety of strategies are available for ligatingfragments of DNA, the choice of which depends on the nature of thetermini of the DNA fragments and which choices can be readily made bythe skilled artisan.

Gene therapy vectors of the present invention, such as the foregoinglentiviral vectors, can be used to express a variety of therapeuticsiRNAs in transformed erythroid cells. In one embodiment, the siRNA ofinterest to be expressed in the vector is derived from a gene that canbe used to treat a hemoglobinopathy, such as an siRNA to BCL11A.

Particular gene therapy constructs of the invention include, but are notlimited to, those shown in FIG. 2. The three lentiviral vectorsdescribed herein are schematically shown with a stem of the shRNAcontaining BCL11A mRNA targeting sequence, while the loop ismiR223-specific. All contain a fluorochrome marker (Venus) and are builtinto a self-inactivating (SIN) delta-U3 LEGO backbone (Ferhse Lab,Germany). A constitutive knock-down lentivirus, where the targetingshRNA is expressed via the very potent, ubiquitously expressed SFFVpromoter, was used to assess functionality and toxicity of the targetingshRNA. An inducible knock-down lentivirus, where the shRNA is expressedvia a PGK tetracycline inducible promoter, was used to assessfunctional, dose- and schedule-dependent effects of the targeting shRNA.A lineage-specific lentivirus, where the shRNA is expressed via aβ-globin LCR promoter landscape (HS2/3 DNA hypersensitive sites, NaldiniLab, Italy) is a therapeutic option to validate in in vivo systems. TheLTR regions further comprise a U3 and U5 region, as well as an R region.The U3 and U5 regions can be modified together or independently tocreate a vector which is self-inactivating, thus increasing the safetyof the vector for use in gene delivery. The U3 and U5 regions canfurther be modified to comprise an insulator element.

The step of facilitating the production of infectious viral particles inthe cells may be carried out using conventional techniques, such asstandard cell culture growth techniques. If desired by the skilledpractitioner, lentiviral stock solutions may be prepared using thevectors and methods of the present invention. Methods of preparing viralstock solutions are known in the art and are illustrated by, e.g., Y.Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau etal. (1992) J. Virol. 66:5110-5113. In the method of producing a stocksolution in the present invention, lentiviral-permissive cells (referredto herein as producer cells) are transfected with the vector system ofthe present invention. The cells are then grown under suitable cellculture conditions, and the lentiviral particles collected from eitherthe cells themselves or from the cell media as described above. Suitableproducer cell lines include, but are not limited to, the human embryonickidney cell line 293, the equine dermis cell line NBL-6, and the caninefetal thymus cell line Cf2TH.

The step of collecting the infectious virus particles also can becarried out using conventional techniques. For example, the infectiousparticles can be collected by cell lysis, or collection of thesupernatant of the cell culture, as is known in the art. Optionally, thecollected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

Other methods relating to the use of viral vectors in gene therapy canbe found in, e.g., Kay, M. A. (1997) Chest 111(6 Supp.):1385-1425;Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory,Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin.Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther.7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M.(1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994)Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opin.Investig. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S.(2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. et al. (2000) Nature408:483-8.

Retroviral vectors, including lentiviral vectors, as described above orcells comprising the same, can be administered in vivo to subjects byany suitable route, as is well known in the art. The term“administration” refers to the route of introduction of a formulatedvector into the body. For example, administration may be intravascular,intraarterial, intravenous, intramuscular, topical, oral, or by gene gunor hypospray instrumentation. Thus, administration can be direct to atarget tissue or through systemic delivery. Administration can be directinjection into the bone marrow. Administration directly to the targettissue can involve needle injection, hypospray, electroporation, or thegene gun. See, e.g., WO 93/18759, which is incorporated by referenceherein.

Alternatively, the retroviral vectors of the invention can beadministered ex vivo or in vitro to cells or tissues using standardtransfection techniques well known in the art.

In one embodiment, the retroviral vectors of the invention can also betransduced into host cells, including embryonic stem cells, somatic stemcells, or progenitor cells. Examples of progenitor host cells which canbe transduced by the retroviral vectors of the invention includeprecursors of erythrocytes and hematopoietic stem cells. In anotherembodiment, the host cell is an erythrocyte. Transduced host cells canbe used as a method of achieving erythroid-specific expression of thegene of interest in the treatment of hemoglobinopathies.

Another aspect of the invention pertains to pharmaceutical compositionsof the lentiviral vectors of the invention. In one embodiment, thecomposition includes a lentiviral vector in a therapeutically effectiveamount sufficient to treat or reduce the risk of developing (e.g.ameliorate the symptoms of a hemoglobinopathy) and a pharmaceuticallyacceptable carrier. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, such as treatment or preventionof a hemoglobinopathic condition. A therapeutically effective amount oflentiviral vector may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thelentiviral vector to elicit a desired response in the individual. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the lentiviral vector are outweighed by thetherapeutically beneficial effects. The potential toxicity of thelentiviral vectors of the invention can be assayed using cell-basedassays or art recognized animal models and a therapeutically effectivemodulator can be selected which does not exhibit significant toxicity.In a preferred embodiment, a therapeutically effective amount of alentiviral vector is sufficient to treat a hemoglobinopathy.

Sterile injectable solutions can be prepared by incorporating lentiviralvector in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens can be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition. In one embodiment, the dosage is ranges from 10³-10⁸ viralparticles/50 kg weight. In other embodiments, the dosage is ranges from10³-10⁵ viral particles/50 kg weight, 10⁴-10⁶ viral particles/50 kgweight, 10⁵-10⁷ viral particles/50 kg weight, 10³-10⁸ viral particles/50kg weight. In one embodiment, the dosage is about 10⁴ viral particles/50kg weight.

The amount of viral vector in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals. However, for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using only routine experimentation.

The present invention contemplates, in particular embodiments, cellsgenetically modified to express the therapeutic polypeptides andinhibitory RNAs contemplated herein, for use in the treatment ofhemoglobinopathies. As used herein, the term “genetically engineered” or“genetically modified” refers to the addition, deletion, or modificationof the genetic material in a cell. The terms, “genetically modifiedcells,” “modified cells,” and, “redirected cells,” are usedinterchangeably. In particular embodiments, cells transduced withvectors contemplated herein are genetically modified. As used herein,the term “gene therapy” refers to the introduction of extra geneticmaterial in the form of DNA or RNA into the total genetic material in acell that restores, corrects, or modifies the cell's physiology toprovide a desired therapeutic outcome.

In various embodiments, the genetically modified cells contemplatedherein are transduced in vitro or ex vivo with vectors of the invention,and optionally expanded ex vivo. The transduced cells are thenadministered to a subject in need of gene therapy.

Cells suitable for transduction and administration in the gene therapymethods contemplated herein include, but are not limited to stem cells,progenitor cells, and differentiated cells. In certain embodiments, thetransduced cells are embryonic stem cells, bone marrow stem cells,umbilical cord stem cells, placental stem cells, mesenchymal stem cells,hematopoietic stem cells, erythroid progenitor cells, and erythroidcells.

Hematopoietic stem cells (HSCs) give rise to committed hematopoieticprogenitor cells (HPCs) that are capable of generating the entirerepertoire of mature blood cells over the lifetime of an organism. Theterm “hematopoietic stem cell” or “HSC” refers to multipotent stem cellsthat give rise to the all the blood cell types of an organism, includingmyeloid (e.g., monocytes and macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells),and lymphoid lineages (e.g., T-cells, B-cells, NK-cells), and othersknown in the art (See Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave,et al., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No.5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al.,U.S. Pat. No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599;Tsukamoto, et al., U.S. Pat. No. 5,716,827). When transplanted intolethally irradiated animals or humans, hematopoietic stem and progenitorcells can repopulate the erythroid, neutrophil-macrophage, megakaryocyteand lymphoid hematopoietic cell pool.

In some embodiments, the transduced cells are hematopoietic stem and/orprogenitor cells isolated from bone marrow, umbilical cord blood, orperipheral circulation. In particular embodiments, the transduced cellsare hematopoietic stem cells isolated from bone marrow, umbilical cordblood, or peripheral circulation.

In one embodiment, the hematopoietic cells are CD34+ cells.

In one embodiment, the hematopoietic cells are erythroid progenitorcells.

In one embodiment, the hematopoietic cells are erythroid cells.

Cells of the invention can be autologous/autogeneic (“self”) ornon-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic).“Autologous,” as used herein, refers to cells from the same subject.“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison. “Syngeneic,” as usedherein, refers to cells of a different subject that are geneticallyidentical to the cell in comparison. “Xenogeneic,” as used herein,refers to cells of a different species to the cell in comparison. Inpreferred embodiments, the cells of the invention are allogeneic. An“isolated cell” refers to a cell that has been obtained from an in vivotissue or organ and is substantially free of extracellular matrix.

Illustrative examples of genetically modified cells suitable forcell-based therapies contemplated herein include, but are not limitedto: embryonic stem cells, bone marrow stem cells, umbilical cord stemcells, placental stem cells, mesenchymal stem cells, hematopoietic stemcells, hematopoietic progenitor cells, myeloid progenitors, erythroidprogenitors, and other erythroid cells.

In preferred embodiments, cells suitable for cell-based therapiescontemplated herein include, but are not limited to: hematopoietic stemor progenitor cells, proerythroblasts, basophilic erythroblasts,polychromatic erythroblasts, orthochromatic erythroblasts, polychromaticerythrocytes, and erythrocytes (RBCs), or any combination thereof.

Methods of Treating, or Reducing a Risk of Developing, aHemoglobinopathy

The present invention provides improved compositions and methods forincreasing HbF production in a cell, by administering vectors thatinhibit expression of BCL11A. The data demonstrate that inhibition ofBCL11A leads to increased expression from the γ-globin genes. Asdisclosed herein, it is an object of the present invention to providecompositions and methods for increasing fetal hemoglobin levels in acell. In some embodiments, the cell is an embryonic stem cell, a somaticstem cell, a progenitor cell, a bone marrow cell, a hematopoietic stemcell, a hematopoietic progenitor cell or a progeny thereof.

Accordingly, one aspect of the invention provides methods for increasingfetal hemoglobin levels expressed by a cell, comprising the steps ofcontacting an embryonic stem cell, a somatic stem cell, a progenitorcell, a bone marrow cell, a hematopoietic stem cell, or a hematopoieticwith an effective amount of a composition comprising at least a virus orvector comprising a nucleic acid molecule described herein, whereby theexpression of BCL11A is reduced and the fetal hemoglobin expression isincreased in the cell, or its progeny, relative to the cell prior tosuch contacting. In one embodiment, the vector or virus expresses an RNAinterference agent which is a BCL11A microRNA which inhibits BCL11A,thereby reducing the expression of BCL11A.

In connection with contacting a cell with an inhibitor of BCL11A,“increasing the fetal hemoglobin levels” in a cell indicates that HbF isat least 5% higher in populations treated with a BCL11A inhibitor, thanin a comparable, control population, wherein no BCL11A inhibitor ispresent. It is preferred that the percentage of HbF expression in aBCL11A inhibitor treated population is at least 10% higher, at least 20%higher, at least 30% higher, at least 40% higher, at least 50% higher,at least 60% higher, at least 70% higher, at least 80% higher, at least90% higher, at least 1-fold higher, at least 2-fold higher, at least5-fold higher, at least 10 fold higher, at least 100 fold higher, atleast 1000-fold higher, or more than a control treated population ofcomparable size and culture conditions. The term “control treatedpopulation” is used herein to describe a population of cells that hasbeen treated with identical media, viral induction, nucleic acidsequences, temperature, confluency, flask size, pH, etc., with theexception of a non-targeting oligonucleotide.

In some embodiments of any of the methods described herein, the subjectis suspected of having, is at risk of having, or has a hemoglobinopathy,e.g., SCD or THAL. It is well within the skills of an ordinarypractitioner to recognize a subject that has, or is at risk ofdeveloping, a hemoglobinopathy.

The subjects can also be those undergoing any of a variety of additionaltherapy treatments. Thus, for example, subjects can be those beingtreated with oxygen, hydroxyurea, folic acid, or a blood transfusion.

Methods of delivering RNA interference agents, e.g., an siRNA, orvectors containing an RNA interference agent, to the target cells, e.g.,erythrocytes or other desired target cells, for uptake include injectionof a composition containing the RNA interference agent, e.g., an siRNA,or directly contacting the cell, e.g., a erythrocyte, with a compositioncomprising an RNA interference agent, e.g., an siRNA. In anotherembodiment, RNA interference agent, e.g., an siRNA may be injecteddirectly into any blood vessel, such as vein, artery, venule orarteriole, via, e.g., hydrodynamic injection or catheterization.Administration may be by a single injection or by two or moreinjections. The RNA interference agent is delivered in apharmaceutically acceptable carrier. One or more RNA interference agentmay be used simultaneously. In one preferred embodiment, only one siRNAthat targets human BCL11A is used. In one embodiment, specific cells aretargeted with RNA interference, limiting potential side effects of RNAinterference caused by non-specific targeting of RNA interference. Themethod can use, for example, a complex or a fusion molecule comprising acell targeting moiety and an RNA interference binding moiety that isused to deliver RNA interference effectively into cells. For example, anantibody-protamine fusion protein when mixed with siRNA, binds siRNA andselectively delivers the siRNA into cells expressing an antigenrecognized by the antibody, resulting in silencing of gene expressiononly in those cells that express the antigen. The siRNA or RNAinterference-inducing molecule binding moiety is a protein or a nucleicacid binding domain or fragment of a protein, and the binding moiety isfused to a portion of the targeting moiety. The location of thetargeting moiety can be either in the carboxyl-terminal oramino-terminal end of the construct or in the middle of the fusionprotein. A viral-mediated delivery mechanism can also be employed todeliver siRNAs to cells in vitro and in vivo as described in Xia, H. etal. (2002) Nat Biotechnol 20(10):1006). Plasmid- or viral-mediateddelivery mechanisms of shRNA may also be employed to deliver shRNAs tocells in vitro and in vivo as described in Rubinson, D. A., et al.((2003) Nat. Genet. 33:401-406) and Stewart, S. A., et al. ((2003) RNA9:493-501). The RNA interference agents, e.g., the siRNAs or shRNAs, canbe introduced along with components that perform one or more of thefollowing activities: enhance uptake of the RNA interfering agents,e.g., siRNA, by the cell, e.g., lymphocytes or other cells, inhibitannealing of single strands, stabilize single strands, or otherwisefacilitate delivery to the target cell and increase inhibition of thetarget gene, e.g., BCL11A. The dose of the particular RNA interferingagent will be in an amount necessary to effect RNA interference, e.g.,post translational gene silencing, of the particular target gene,thereby leading to inhibition of target gene expression or inhibition ofactivity or level of the protein encoded by the target gene.

In one embodiment of any methods described herein, the embryonic stemcell, somatic stem cell, progenitor cell, bone marrow cell, orhematopoietic progenitor cell, or hematopoietic stem cell (HSC) iscontacted ex vivo or in vitro. In a specific embodiment, the cell beingcontacted is a cell of the erythroid lineage. In one embodiment, thecomposition inhibits BCL11A expression.

In one embodiment of any methods described herein, the embryonic stemcell, somatic stem cell, progenitor cell, bone marrow cell, orhematopoietic progenitor cell, or HSC is isolated from the subject priorto contacting with the composition described herein or contacting withthe virus or vector carrying a nucleic acid molecule comprising anucleic acid sequence selected from a group consisting of SEQ IDNOS:1-10,13-18, 25-44, or contacting with the virus or vector expressinga synthetic BCL11A microRNA described herein.

Mature blood cells have a finite lifespan and must be continuouslyreplaced throughout life. Blood cells are produced by the proliferationand differentiation of a very small population of pluripotenthematopoietic stem cells (HSCs) that also have the ability to replenishthemselves by self-renewal. HSCs are multipotent, self-renewingprogenitor cells that develop from mesodermal hemangioblast cells. HSCsare the blood cells that give rise to all the other blood cells, thatincludes all the differentiated blood cells from the erythroid, lymphoidand myeloid lineages. HSCs are located in the adult bone marrow,peripheral blood, and umbilical cord blood.

During differentiation, the progeny of HSCs progress through variousintermediate maturational stages, generating multi-potentialhematopoietic progenitor cells and lineage-committed hematopoieticprogenitor cells, prior to reaching maturity. Bone marrow (BM) is themajor site of hematopoiesis in humans and, under normal conditions, onlysmall numbers of HSCs and hematopoietic progenitor cells can be found inthe peripheral blood (PB). Treatment with cytokines (in particulargranulocyte colony-stimulating factor; G-CSF), myelosuppressive drugsused in cancer treatment, and compounds that disrupt the interactionbetween hematopoietic cells and BM stromal cells can rapidly mobilizelarge numbers of stem and progenitor cells into the circulation.

“Hematopoietic progenitor cell” as the term is used herein, refers tocells of a hematopoietic stem cell lineage that give rise to all theblood cell types including the myeloid (monocytes and macrophages,neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and the lymphoid lineages(T-cells, B-cells, NK-cells). A “cell of the erythroid lineage”indicates that the cell being contacted is a cell that undergoeserythropoeisis such that upon final differentiation it forms anerythrocyte or red blood cell (RBC). Such cells belong to one of threelineages, erythroid, lymphoid, and myeloid, originating from bone marrowhematopoietic progenitor cells. Upon exposure to specific growth factorsand other components of the hematopoietic microenvironment,hematopoietic progenitor cells can mature through a series ofintermediate differentiation cellular types, all intermediates of theerythroid lineage, into RBCs. Thus, cells of the “erythroid lineage,” asthe term is used herein, comprise hematopoietic progenitor cells,rubriblasts, prorubricytes, erythroblasts, metarubricytes,reticulocytes, and erythrocytes.

In some embodiment of any methods described herein, the hematopoieticprogenitor cell has at least one of the cell surface markercharacteristic of hematopoietic progenitor cells: CD34+, CD59+,Thy1/CD90+, CD38^(lo/−), and C-kit/CD117+. Preferably, the hematopoieticprogenitor cells have several of these markers.

In some embodiment of any methods described herein, the hematopoieticprogenitor cells of the erythroid lineage have the cell surface markercharacteristic of the erythroid lineage: CD71 and Ter119.

In some embodiment of any methods described herein, the HSC has at leastone of the cell surface marker characteristic of hematopoieticprogenitor cells: CD34+, CD59+, Thy1/CD90+, CD38^(lo/−), andC-kit/CD117+.

The HSCs, similar to the hematopoietic progenitor cells, are capable ofproliferation and giving rise to more progenitor cells having theability to generate a large number of mother cells that can in turn giverise to differentiated or differentiable daughter cells. The daughtercells themselves can be induced to proliferate and produce progeny thatsubsequently differentiate into one or more mature cell types, whilealso retaining one or more cells with parental developmental potential.The term “stem cell” refers then, to a cell with the capacity orpotential, under particular circumstances, to differentiate to a morespecialized or differentiated phenotype, and which retains the capacity,under certain circumstances, to proliferate without substantiallydifferentiating. In one embodiment, the term progenitor or stem cellrefers to a generalized mother cell whose descendants (progeny)specialize, often in different directions, by differentiation, e.g., byacquiring completely individual characters, as occurs in progressivediversification of embryonic cells and tissues. Cellular differentiationis a complex process typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell which itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typeseach can give rise to may vary considerably. Some differentiated cellsalso have the capacity to give rise to cells of greater developmentalpotential. Such capacity may be natural or may be induced artificiallyupon treatment with various factors. In many biological instances, stemcells are also “multipotent” because they can produce progeny of morethan one distinct cell type, but this is not required for “stem-ness.”Self-renewal is the other classical part of the stem cell definition,and it is essential as used in this document. In theory, self-renewalcan occur by either of two major mechanisms. Stem cells may divideasymmetrically, with one daughter retaining the stem state and the otherdaughter expressing some distinct other specific function and phenotype.Alternatively, some of the stem cells in a population can dividesymmetrically into two stems, thus maintaining some stem cells in thepopulation as a whole, while other cells in the population give rise todifferentiated progeny only. Generally, “progenitor cells” have acellular phenotype that is more primitive (i.e., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell). Often, progenitor cells also have significant orvery high proliferative potential. Progenitor cells can give rise tomultiple distinct differentiated cell types or to a singledifferentiated cell type, depending on the developmental pathway and onthe environment in which the cells develop and differentiate.

In one embodiment of any methods described herein, the hematopoieticstem cell or hematopoietic progenitor cell is collected from peripheralblood, cord blood, chorionic villi, amniotic fluid, placental blood, orbone marrow.

In one embodiment of any methods described herein, the embryonic stemcell, somatic stem cell, progenitor cell, or bone marrow cell iscollected from peripheral blood, cord blood, chorionic villi, amnioticfluid, placental blood, or bone marrow.

Peripheral blood progenitor cells (PBPC) have become the preferredsource of hematopoetic progenitor cells for allogeneic and autologoustransplantation because of technical ease of collection and shorter timerequired for engraftment. Traditionally, granulocyte-colony stimulatingfactor (G-CSF) has been used to stimulate more PBPC and release ofhematopoetic progenitor cells from the bone marrow. Although regimensusing G-CSF usually succeed in collecting adequate numbers of PBPC fromhealthy donors, 5%-10% will mobilize stem cells poorly and may requiremultiple large volume apheresis or bone marrow harvesting.

In some embodiments of any methods described herein, the embryonic stemcell, somatic stem cell, progenitor cell, bone marrow cell, orhematopoietic progenitor cell, or HSC is selected for the CD34+ surfacemarker prior to the contacting.

Accordingly, in one embodiment of any methods described herein, theisolated CD34+ embryonic stem cell, isolated CD34+ somatic stem cell,isolated CD34+ progenitor cell, isolated CD34+ bone marrow cell,isolated CD34+ hematopoietic progenitor cell, or isolated CD34+ HSC iscontacted with the composition described herein or contacted with thevirus or vector carrying a nucleic acid molecule comprising a nucleicacid sequence selected from a group consisting of SEQ ID NOS:1-10,13-18,25-44, or contacted with the virus or vector expressing a syntheticBCL11A microRNA described herein.

In one embodiment of any methods described herein, the embryonic stemcell, somatic stem cell, progenitor cell, bone marrow cell, orhematopoietic progenitor cell, or HSC is cryopreserved prior to anycontacting with the composition described herein or contacting with thevirus or vector carrying a nucleic acid molecule comprising a nucleicacid sequence selected from a group consisting of SEQ ID NOS:1-10,13-18,25-44, or contacting with the virus or vector expressing a syntheticBCL11A microRNA described herein.

In one embodiment of any methods described herein, the contacting is invitro, ex vivo or in vivo.

In one embodiment of any methods described herein, the contacting isrepeated at least once. That is, after the initial first contacting ofthe embryonic stem cell, somatic stem cell, progenitor cell, bone marrowcell, or hematopoietic progenitor cell, or HSC with the compositiondescribed herein or contacting with the virus or vector carrying anucleic acid molecule comprising a nucleic acid sequence selected from agroup consisting of SEQ ID NOS:1-10,13-18, 25-44, or contacting with thevirus or vector expressing a synthetic BCL11A microRNA described herein,the cell is washed, and the washed cell is then contacted for a secondtime with the composition described herein or contacted with the virusor vector carrying a nucleic acid molecule comprising a nucleic acidsequence selected from a group consisting of SEQ ID NOS:1-10,13-18,25-44, or contacted with the virus or vector expressing a syntheticBCL11A microRNA described herein.

In other embodiments, the contacting is repeated at least twice afterthe initial first contacting.

In one embodiment of any methods described herein, after the contacting,the contacted embryonic stem cell, somatic stem cell, progenitor cell,bone marrow cell, or hematopoietic progenitor cell, or HSC iscryopreserved prior to use, for example, ex vivo expansion and/orimplantation into a subject.

In one embodiment of any methods described herein, after the contacting,the contacted embryonic stem cell, somatic stem cell, progenitor cell,bone marrow cell, or hematopoietic progenitor cell, or HSC is cultureexpanded ex vivo prior to use, for example, cryopreservation, and/orimplantation/engraftment into a subject.

In one embodiment of any methods described herein, after the contacting,the contacted embryonic stem cell, somatic stem cell, progenitor cell,bone marrow cell, or hematopoietic progenitor cell, or HSC isdifferentiated in culture ex vivo prior to use, for example,cryopreservation, and/or implantation/engraftment into a subject.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a hematopoietic progenitorcell), which in turn can differentiate into other types of precursorcells further down the pathway (such as an erthyrocyte precursor), andthen to an end-stage differentiated cell, such as an erthyrocyte, whichplays a characteristic role in a certain tissue type, and may or may notretain the capacity to proliferate further.

In one embodiment, the inhibitor of BCL11A expression is a BCL11Aspecific RNA interference agent, or a vector encoding said BCL11Aspecific RNA interference agent. In one specific embodiment, the RNAinterference agent comprises one or more of the nucleotide sequences ofSEQ ID NOS:1-10,13-18, 25-44.

A “nucleic acid,” as described herein, can be RNA or DNA, and can besingle or double stranded, and can be selected, for example, from agroup including: nucleic acid encoding a protein of interest,oligonucleotides, nucleic acid analogues, for example peptide-nucleicacid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid(LNA). Such nucleic acid sequences include, for example, but are notlimited to, nucleic acid sequence encoding proteins, for example thatact as transcriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but are not limited toRNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.

As disclosed herein, it is an object of the present invention to providea method for increasing fetal hemoglobin levels in a subject.

Accordingly, one aspect of the present invention provides a method forincreasing fetal hemoglobin levels in a subject in need thereof, themethod comprising the step of contacting a hematopoietic progenitor cellor a HSC in the subject with an effective amount of a compositioncomprising an inhibitor of BCL11A, whereby HbF expression is increased,relative to expression prior to such contacting. In one embodiment, theinhibitor of BCL11A is an RNA interference agent which comprises one ormore of the nucleotide sequences of SEQ ID NOS:1-10,13-18, 25-44, or asynthetic BCL11A microRNA described herein.

In connection with contacting a cell in a subject with an inhibitor ofBCL11A, “increasing HbF levels in a subject” indicates that HbF in thesubject is at least 5% higher in populations treated with a BCL11Ainhibitor, than a comparable, control population, wherein no BCL11Ainhibitor is present. It is preferred that the fetal hemoglobinexpression in a BCL11A inhibitor treated subject is at least 10% higher,at least 20% higher, at least 30% higher, at least 40% higher, at least50% higher, at least 60% higher, at least 70% higher, at least 80%higher, at least 90% higher, at least 1-fold higher, at least 2-foldhigher, at least 5-fold higher, at least 10 fold higher, at least 100fold higher, at least 1000-fold higher, or more than a comparablecontrol treated subject. The term “comparable control treated subject”is used herein to describe a subject that has been treated identically,with the exception of the addition of a non-targeting oligonucleotide.

Accordingly, in one embodiment, the subject has been diagnosed with ahemoglobinopathy. In a further embodiment, the hemoglobinopathy is aSCD. As used herein, SCD can be sickle cell anemia, sickle-hemoglobin Cdisease (HbSC), sickle beta-plus-thalassemia (HbS/β+), or sicklebeta-zero-thalassemia (HbS/β0). In another preferred embodiment, thehemoglobinopathy is THAL.

The treatment according to the present invention ameliorates one or moresymptoms associated with the disorder by increasing the amount of fetalhemoglobin in the individual. Symptoms typically associated with ahemoglobinopathy, include for example, anemia, tissue hypoxia, organdysfunction, abnormal hematocrit values, ineffective erythropoiesis,abnormal reticulocyte (erythrocyte) count, abnormal iron load, thepresence of ring sideroblasts, splenomegaly, hepatomegaly, impairedperipheral blood flow, dyspnea, increased hemolysis, jaundice, anemicpain crises, acute chest syndrome, splenic sequestration, priapism,stroke, hand-foot syndrome, and pain such as angina pectoris.

In one embodiment, the hematopoietic progenitor cell or HSC is contactedex vivo or in vitro, and the cell or its progeny is administered to thesubject. In a further embodiment, the hematopoietic progenitor cell is acell of the erythroid lineage.

In one embodiment, the hematopoietic progenitor cell or HSC is contactedwith a composition comprising of an inhibitor of BCL11A and apharmaceutically acceptable carrier or diluent. In one embodiment, thecomposition is administered by injection, infusion, instillation, oringestion. In one embodiment, the composition is administered by directinjection into the bone marrow.

In one embodiment of any one method described, the gene therapy methodis used to treat, prevent, or ameliorate a hemoglobinopathy is selectedfrom the group consisting of: hemoglobin C disease, hemoglobin sicklecell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia,β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemia,and hemoglobin H disease.

In various embodiments of any one method described, the retroviralvectors are administered by direct injection to a cell, tissue, or organof a subject in need of gene therapy, in vivo. In various otherembodiments of any one method described, cells are transduced in vitroor ex vivo with vectors of the invention, and optionally expanded exvivo. The transduced cells are then administered to a subject in need ofgene therapy.

A “subject,” as used herein, includes any animal that exhibits a symptomof a monogenic disease, disorder, or condition that can be treated withthe gene therapy vectors, cell-based therapeutics, and methods disclosedelsewhere herein. In preferred embodiments, a subject includes anyanimal that exhibits symptoms of a disease, disorder, or condition ofthe hematopoietic system, e.g., a hemoglobinopathy, that can be treatedwith the gene therapy vectors, cell-based therapeutics, and methodscontemplated herein. Suitable subjects (e.g., patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, preferably, human patients, are included. Typical subjectsinclude animals that exhibit aberrant amounts (lower or higher amountsthan a “normal” or “healthy” subject) of one or more physiologicalactivities that can be modulated by gene therapy.

In one embodiment, as used herein “treatment” or “treating,” includesany beneficial or desirable effect on the symptoms or pathology of adisease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated. In another embodiment, treatment can involve optionallyeither the reduction or amelioration of symptoms of the disease orcondition, or the delaying of the progression of the disease orcondition. “Treatment” does not necessarily indicate completeeradication or cure of the disease or condition, or associated symptomsthereof.

In one embodiment, as used herein, “prevent,” and similar words such as“prevented,” “preventing” etc., indicate an approach for preventing,inhibiting, or reducing the likelihood of the occurrence or recurrenceof, a disease or condition. In another embodiment, the term refers todelaying the onset or recurrence of a disease or condition or delayingthe occurrence or recurrence of the symptoms of a disease or condition.In another embodiment, as used herein, “prevention” and similar wordsincludes reducing the intensity, effect, symptoms and/or burden of adisease or condition prior to onset or recurrence of the disease orcondition.

In one embodiment of any one method described, the method furthercomprises selecting a subject in need of the gene therapy described. Forexample, a subject exhibiting symptoms or cytology of a hemoglobinopathyis selected from the group consisting of hemoglobin C disease,hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditaryanemia, thalassemia, β-thalassemia, thalassemia major, thalassemiaintermedia, α-thalassemia, and hemoglobin H disease. Alternatively, thesubject carrys a genetic mutation that is associated with ahemoglobinopathy, a genetic mutation described herein. For example, asubject diagnosis of SCD with genotype HbSS, HbS/β0 thalassemia, HbSD,or HbSO, and/or with HbF<10% by electrophoresis.

In various embodiments of any one method described, a subject in need ofgene therapy is administered a population of cells comprising aneffective amount of genetically modified cells contemplated herein. Thatis a genetically modified cells that express one or more of thenucleotide sequences of SEQ ID NOS:1-10,13-18, 25-44, or a syntheticBCL11A microRNA described herein.

As used herein, the term “amount” refers to “an amount effective” or “aneffective amount” of a virus or transduced therapeutic cell to achieve abeneficial or desired prophylactic or therapeutic result, includingclinical results.

A “prophylactically effective amount” refers to an amount of a virus ortransduced therapeutic cell effective to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount is less than thetherapeutically effective amount.

A “therapeutically effective amount” of a virus or transducedtherapeutic cell may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thestem and progenitor cells to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the virus or transduced therapeuticcells are outweighed by the therapeutically beneficial effects. The term“therapeutically effective amount” includes an amount that is effectiveto “treat” a subject (e.g., a patient).

In one embodiment, the present invention provides a method of providinga transduced cell to a subject that comprises administering, e.g.,parenterally, one or more cells transduced with a vector contemplatedherein into the subject. In one embodiment, the vector is one thatcarrys one or more of the nucleotide sequences of SEQ ID NOS:1-10,13-18,25-44, or a synthetic BCL11A microRNA described herein.

In a particular embodiment, a method of preventing, ameliorating, ortreating a hemoglobinopathy in a subject is provided. The methodcomprises administering a population of cells comprising hematopoieticcells transduced with a vector contemplated herein. In one embodiment,the vector is one that carrys one or more of the nucleotide sequences ofSEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA describedherein.

In particular embodiments, a population of cells administered to asubject comprises hematopoietic stem or progenitor cells,proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts,orthochromatic erythroblasts, polychromatic erythrocytes, anderythrocytes (RBCs), or any combination thereof, and any proportion ofwhich may be genetically modified by the vectors contemplated herein. Inone embodiment, the vector is one that carrys one or more of thenucleotide sequences of SEQ ID NOS:1-10,13-18, 25-44, or a syntheticBCL11A microRNA described herein.

The genetically modified cells may be administered as part of a bonemarrow or cord blood transplant in an individual that has or has notundergone bone marrow ablative therapy. In one embodiment, geneticallymodified cells contemplated herein are administered in a bone marrowtransplant to an individual that has undergone chemoablative orradioablative bone marrow therapy.

In one embodiment, a dose of genetically modified cells is delivered toa subject intravenously. In one embodiment, genetically modifiedhematopoietic cells are intravenously administered to a subject.

In particular embodiments, patients receive a dose of geneticallymodified cells, e.g., hematopoietic stem cells, of about 1×10⁵ cells/kg,about 5×10⁵ cells/kg, about 1×10⁶ cells/kg, about 2×10⁶ cells/kg, about3×10⁶ cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about 6×10⁶cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, about 1×10⁷ cells/kg, about 5×10⁷ cells/kg, about 1×10⁸cells/kg, or more in one single intravenous dose. In certainembodiments, patients receive a dose of genetically modified cells,e.g., hematopoietic stem cells, of at least 1×10⁵ cells/kg, at least5×10⁵ cells/kg, at least 1×10⁶ cells/kg, at least 2×10⁶ cells/kg, atleast 3×10⁶ cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg,at least 6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶cells/kg, at least 9×10⁶ cells/kg, at least 1×10⁷ cells/kg, at least5×10⁷ cells/kg, at least 1×10⁸ cells/kg, or more in one singleintravenous dose.

In an additional embodiment, patients receive a dose of geneticallymodified cells, e.g., hematopoietic stem cells, of about 1×10⁵ cells/kgto about 1×10⁸ cells/kg, about 1×10⁶ cells/kg to about 1×10⁸ cells/kg,about 1×10⁶ cells/kg to about 9×10⁶ cells/kg, about 2×10⁶ cells/kg toabout 8×10⁶ cells/kg, about 2×10⁶ cells/kg to about 8×10⁶ cells/kg,about 2×10⁶ cells/kg to about 5×10⁶ cells/kg, about 3×10⁶ cells/kg toabout 5×10⁶ cells/kg, about 3×10⁶ cells/kg to about 4×10⁸ cells/kg, orany intervening dose of cells/kg.

In various embodiments, the methods of the invention provide more robustand safe gene therapy than existing methods and comprise administering apopulation or dose of cells comprising about 5% transduced cells, about10% transduced cells, about 15% transduced cells, about 20% transducedcells, about 25% transduced cells, about 30% transduced cells, about 35%transduced cells, about 40% transduced cells, about 45% transducedcells, or about 50% transduced cells, to a subject.

In one embodiment, the invention provides genetically modified cells,such as a stem cell, e.g., hematopoietic stem cell, with the potentialto expand or increase a population of erythroid cells. In particularembodiments, hematopoietic stem cells are transduced with a vector ofthe invention and administered to an individual in need of therapy forhemoglobinopathy. Hematopoietic stem cells are the origin of erythroidcells and thus, are preferred. In one embodiment, the vector is one thatcarrys one or more of the nucleotide sequences of SEQ ID NOS:1-10,13-18,25-44, or a synthetic BCL11A microRNA described herein.

As used herein, the term “pharmaceutically acceptable,” and grammaticalvariations thereof, as they refer to compositions, carriers, diluentsand reagents, are used interchangeably and represent that the materialsare capable of administration to or upon a subject without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset, and the like. Each carrier must also be“acceptable” in the sense of being compatible with the other ingredientsof the formulation. A pharmaceutically acceptable carrier will notpromote the raising of an immune response to an agent with which it isadmixed, unless so desired. The preparation of a pharmacologicalcomposition that contains active ingredients dissolved or dispersedtherein is well understood in the art and need not be limited based onformulation. The pharmaceutical formulation contains a compound of theinvention in combination with one or more pharmaceutically acceptableingredients. The carrier can be in the form of a solid, semi-solid orliquid diluent, cream or a capsule. Typically such compositions areprepared as injectable either as liquid solutions or suspensions,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe present invention can include pharmaceutically acceptable salts ofthe components therein. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of thepolypeptide) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic, and the like. Salts formed with the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like. Physiologically tolerable carriers arewell known in the art. Exemplary liquid carriers are sterile aqueoussolutions that contain no materials in addition to the activeingredients and water, or contain a buffer such as sodium phosphate atphysiological pH value, physiological saline or both, such asphosphate-buffered saline. Still further, aqueous carriers can containmore than one buffer salt, as well as salts such as sodium and potassiumchlorides, dextrose, polyethylene glycol and other solutes. Liquidcompositions can also contain liquid phases in addition to and to theexclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the invention that willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. The phrase “pharmaceutically acceptablecarrier or diluent” means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body.

In one embodiment of any methods described, as used herein,“administered” refers to the placement of an inhibitor of BCL11A into asubject by a method or route which results in at least partiallocalization of the inhibitor at a desired site. An agent which inhibitsBCL11A can be administered by any appropriate route which results ineffective treatment in the subject, i.e., administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e., at least one agent, which inhibitsBCL11A, is active in the desired site for a period of time. The periodof time the inhibitor is active depends on the half-life in vivo afteradministration to a subject, and can be as short as a few hours, e.g.,at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours,at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours,at least 12 hours, at least 18 hours, at least 24 hours, to a few days,to as long as several years. Modes of administration include injection,infusion, instillation, or ingestion. “Injection” includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

In one embodiment, the composition described herein, or the virus orvector carrying a nucleic acid molecule comprising a nucleic acidsequence selected from a group consisting of SEQ ID NOS:1-10,13-18,25-44, or the virus or vector expressing a synthetic BCL11A microRNAdescribed herein, is injected into the bone marrow.

In one embodiment, the hematopoietic progenitor cell or HSC from asubject needing treatment is contacted with a composition that inhibitsBCL11A expression. In other embodiments, the composition comprises avirus or vector carrying a nucleic acid molecule comprising a nucleicacid sequence selected from a group consisting of SEQ ID NOS:1-10,13-18,25-44, or a virus or vector expressing a synthetic BCL11A microRNAdescribed herein. The subject needing treatment is one diagnosed with ahemoglobinopathy such as SCD or THAL.

By “inhibits BCL11A expression” is meant that the amount of expressionof BCL11A is at least 5% lower in populations treated with a BCL11Ainhibitor, than a comparable, control population, wherein no BCL11Ainhibitor is present. It is preferred that the percentage of BCL11Aexpression in a BCL11A inhibitor treated population is at least 10%lower, at least 20% lower, at least 30% lower, at least 40% lower, atleast 50% lower, at least 60% lower, at least 70% lower, at least 80%lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower,at least 5-fold lower, at least 10 fold lower, at least 100 fold lower,at least 1000-fold lower, or more than a comparable control treatedpopulation in which no BCL11A inhibitor is added.

In one embodiment, the nucleic acid is a BCL11A specific RNAinterference agent or a vector encoding the RNA interference agent. Inone embodiment, the RNA interference agent comprises one or more of thenucleotide sequences of SEQ ID NOs:1-10,13-18, 25-44.

As an example of a method of treatment of a subject or reducing the riskof developing a hemoglobinopathy in a subject, the method comprisesadministering to the subject a composition comprising modifiedengineered cells that comprise a vector carrying a nucleic acid sequenceselected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-44,or a BCL11A microRNA described herein. In one embodiment, the methodfurther comprises identifying a subject having a hemoglobinopathy or isat risk of developing a hemoglobinopathy. In another embodiment, themethod further comprises selecting the identified subject having ahemoglobinopathy or is at risk of developing a hemoglobinopathy.

As another example of a method of treatment of a subject or reducing therisk of developing a hemoglobinopathy in a subject, the method comprisesthe following steps: mobilize the hematopoietic stem and hematopoieticprogenitor cells in a subject; harvest and collect peripheral blood fromthe subject, positive selection of CD34+ cells from the peripheralblood, transduce or transfect the CD34+ selected cells in vitro with avector carrying a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNAdescribed herein; wash the transduced CD34+ selected cells; andadminister the cells into the subject. In one embodiment, the methodfurther comprises identifying a subject having a hemoglobinopathy or isat risk of developing a hemoglobinopathy. In one embodiment, the methodfurther comprises selecting the subject having a hemoglobinopathy or isat risk of developing a hemoglobinopathy. In another embodiment, themethod further comprises expanding in culture the washed, transducedCD34+ selected cells in vitro prior to administering to the subject. Inanother embodiment, the method further comprises differentiating inculture the washed, transduced CD34+ selected cells in vitro prior toadministering to the subject.

As another example of a method of treatment of a subject or reducing therisk of developing a hemoglobinopathy in a subject, the method comprisesthe following steps: mobilize the hematopoietic stem and hematopoieticprogenitor cells in a donor subject; harvest and collect peripheralblood from the donor subject, positive selection of CD34+ cells from theperipheral blood, transduce or transfect the CD34+ selected cells invitro with a vector carrying a nucleic acid sequence selected from thegroup consisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11AmicroRNA described herein; wash the transduced CD34+ selected cells; andadminister the cells into a recipient subject. In one embodiment, themethod further comprises selecting a recipient subject having ahemoglobinopathy or is at risk of developing a hemoglobinopathy. Inanother embodiment, the method further comprises expanding in culturethe washed, transduced CD34+ selected cells in vitro prior toadministering to the recipient subject. In another embodiment, themethod further comprises differentiating in culture the washed,transduced CD34+ selected cells in vitro prior to administering to therecipient subject.

As another example of a method of treatment of a subject or reducing therisk of developing a hemoglobinopathy in a subject, the method comprisesthe following steps: harvest and collect the blood from the bone marrowof a subject, positive selection of CD34+ cells from the bone marrowblood, transduce or transfect the CD34+ selected cells in vitro with avector carrying a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNAdescribed herein; wash the transduced CD34+ selected cells; andadminister the cells into the subject. In one embodiment, the methodfurther comprises identifying a subject having a hemoglobinopathy or isat risk of developing a hemoglobinopathy. In one embodiment, the methodfurther comprises selecting the subject having a hemoglobinopathy or isat risk of developing a hemoglobinopathy. In another embodiment, themethod further comprises expanding in culture the washed, transducedCD34+ selected cells in vitro prior to administering to the subject. Inanother embodiment, the method further comprises differentiating inculture the washed, transduced CD34+ selected cells in vitro prior toadministering to the subject.

As another example of a method of treatment of a subject or reducing therisk of developing a hemoglobinopathy in a subject, the method comprisesthe following steps: harvest and collect the blood from the bone marrowof a donor subject, positive selection of CD34+ cells from the bonemarrow blood, transduce or transfect the CD34+ selected cells in vitrowith a vector carrying a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNAdescribed herein; wash the transduced CD34+ selected cells; andadminister the cells into a recipient subject. In one embodiment, themethod further comprises identifying a recipient subject having ahemoglobinopathy or is at risk of developing a hemoglobinopathy. In oneembodiment, the method further comprises selecting a recipient subjecthaving a hemoglobinopathy or is at risk of developing ahemoglobinopathy. In another embodiment, the method further comprisesexpanding in culture the washed, transduced CD34+ selected cells invitro prior to administering to the recipient subject. In anotherembodiment, the method further comprises differentiating in culture thewashed, transduced CD34+ selected cells in vitro prior to administeringto the recipient subject.

In one embodiment, the disclosure herein provides a modified engineeredcell comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNAdescribed herein.

In one embodiment, the disclosure herein provides a modified engineeredcell that has been transduced or transfected with a vector comprising anucleic acid sequence selected from the group consisting of SEQ IDNOS:1-10, 13-18 and 25-44, or a BCL11A microRNA described herein. In oneembodiment, the vector is a lentivirus.

In one embodiment, the disclosure herein provides a method of treatmentof a subject or reducing the risk of developing a hemoglobinopathy in asubject, the method comprises administering a modified engineered cellthat has been transduced or transfected with a vector comprising anucleic acid sequence selected from the group consisting of SEQ IDNOS:1-10, 13-18 and 25-44, or a BCL11A microRNA described herein. In oneembodiment, the vector is a lentivirus.

In one embodiment, the disclosure herein provides a method of treatmentof a subject or reducing the risk of developing a hemoglobinopathy in asubject, the method comprises administering a modified engineered cellcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNA described herein.

In one embodiment, the modified engineered cell is an embryonic stemcell, a somatic stem cell, a progenitor cell, a bone marrow cell, ahematopoietic stem cell, or a hematopoietic progenitor cell.

In one embodiment, the modified engineered cell is a cell that has beendifferentiated from an embryonic stem cell, a somatic stem cell, aprogenitor cell, a bone marrow cell, a hematopoietic stem cell, or ahematopoietic progenitor cell.

In one embodiment, the modified engineered cell is a cell that has beendifferentiated into the erythroid lineage.

In one embodiment, the modified engineered cell is a cell that has beendifferentiated into an erythrocyte.

In one embodiment, the modified engineered cell is a CD34+ cell.

The present invention can be defined in any of the following numberedparagraphs.

-   -   [1] A synthetic BCL11A microRNA comprising a first BCL11A        segment, a loop segment; and a second BCL11A segment arranged in        tandem in a 5′ to 3′ direction, wherein the loop segment is        between and directly linked to the first and second BCL11A        segments, and wherein the second BCL11A segment is complementary        to the first BCL11A segment so that the first and second BCL11A        segments base pair to form a hairpin loop with the loop segment        forming the loop portion of the hairpin loop thus formed.    -   [2] The synthetic BCL11A microRNA of paragraph 1, wherein the        first and second BCL11A segments are about 18 to 25 nucleotides        long.    -   [3] The synthetic BCL11A microRNA of paragraph 1 or 2, wherein        the first BCL11A segment contains a sequence derived from a        BCL11A mRNA sequence.    -   [4] The synthetic BCL11A microRNA of any one of paragraphs 1-3,        wherein the first BCL11A segment is complementary to the second        BCL11A segment.    -   [5] The synthetic BCL11A microRNA of any one of paragraphs 1-4,        wherein the first BCL11A segment starts with a -GCGC- at the 5′        end and the second BCL11A segment ends with a -GCGC- at the 3′        end.    -   [6] The synthetic BCL11A microRNA of any one of paragraphs 1-5,        wherein the first BCL11A segment is selected from the group        consisting of CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; derived        from BCL11A miR1 oligo described herein), CCAGAGGATGACGATTGTTTA        (SEQ. ID. NO: 47; derived from BCL11A miR2 oligo described        herein), TCGGAGACTCCAGACAATCGC (SEQ. ID. NO: 48; derived from        BCL11A E3 oligo or shRNA1 or E3 described herein),        CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 or        B5 described herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50;        derived from shRNA4 or B11 described herein),        TTCTCTTGCAACACGCACAGA (SEQ. ID. NO: 51; derived from BCL11A D8        oligo or shRNA3 or D8 described herein), GATCGAGTGTTGAATAATGAT        (SEQ. ID. NO: 52; derived from shRNA5 or 50D12 o1 D12 described        herein), CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53; derived from        shRNA5 or 50A5 described herein), CACTGTCCACAGGAGAAGCCA (SEQ.        ID. NO: 54; derived from shRNA7 or 50B11 described herein),        ACAGTACCCTGGAGAAACACA (SEQ. ID. NO: 55; derived from BCL11A        XLC4, shRNA8 and 50C4 described herein), CAACAAGATGAAGAGCACCAA        (SEQ. ID. NO: 56; derived from BCL11A Non-targeting oligos        described herein), gcgcCGCACAGAACACTCATG (SEQ. ID. NO: 57;        derived from miR1G5 oligo described herein),        GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58; derived from E3G5 or E3        mod oligo or shRNA1 mod described herein), gcgcCCTCCAGGCAGCTCAAA        (SEQ. ID. NO: 59; derived from B5G5 or shRNA2 mod described        herein); gcgcTCAGGACTAGGTGCAGA (SEQ. ID. NO: 60; derived from        B11G5 or shRNA4 mod described herein); gcgcGATCGAGTGTTGAATAA        (SEQ. ID. NO: 61; derived from 50D12G5, D12G4 or shRNA5 mod        described herein); gcgcCAGTACCCTGGAGAAAC (SEQ. ID. NO: 62;        derived from 50A5G5 or shRNA6 mod described herein);        gcgcCACTGTCCACAGGAGAA (SEQ. ID. NO: 63; derived from 50B11G5 or        shRNA7 mod described herein); GCGCTTCTCTTGCAACACGCA (SEQ. ID.        NO: 64; derived from BCL11A D8G5 or D8 mod or shRNA3 mod        described herein), GCGCACAGTACCCTGGAGAAA (SEQ. ID. NO: 65;        derived from BCL11A C4G5, or C4 mod or shRNA8 mod described        herein), CGCACAGAACACTCATGGATT (SEQ. ID. NO: 66; derived from        BCL11A D12G5-2 described herein), and ACGCTCGCACAGAACACTCATGGATT        (SEQ. ID. NO: 67; derived from BCL11A D12G5-2 described herein).    -   [7] The synthetic BCL11A microRNA of any one of paragraphs 1-6,        wherein the loop segment is derived from a microRNA.    -   [8] The synthetic BCL11A microRNA of paragraph 7, wherein the        microRNA is a hematopoietic specific microRNA.    -   [9] The synthetic BCL11A microRNA of paragraph 8, wherein the        microRNA is miR223.    -   [10] The synthetic BCL11A microRNA of paragraph 9, wherein the        loop segment is ctccatgtggtagag (SEQ ID NO: 68).    -   [11] The synthetic BCL11A microRNA of any one of paragraphs        1-10, wherein the microRNA comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOS:1-10, 13-18 and        25-44.    -   [12] A method of treating, or reducing a risk of developing, a        hemoglobinopathy in a subject, the method comprising expressing        in vivo at least one synthetic BCL11A microRNA of any one of        paragraphs 1-11 in the subject.    -   [13] The method of paragraph 12, wherein the in vivo expression        occurs in an embryonic stem cell, a somatic stem cell, a        progenitor cell, a bone marrow cell, a hematopoietic stem cell,        or a hematopoietic progenitor cell in the subject.    -   [14] A method of treating, or reducing a risk of developing, a        hemoglobinopathy in a subject, the method comprising expressing        at least one synthetic BCL11A microRNA of any one paragraphs        1-11 in an embryonic stem cell, a somatic stem cell, a        progenitor cell, a bone marrow cell, a hematopoietic stem cell,        or a hematopoietic progenitor cell of the subject wherein the        expression is ex vivo, and implanting the cell into the subject.    -   [15] A method of increasing fetal hemoglobin levels expressed by        a cell comprising expressing at least one synthetic BCL11A        microRNA of any one paragraphs 1-11 in a cell, wherein the cell        is an embryonic stem cell, a somatic stem cell, a progenitor        cell, a bone marrow cell, a hematopoietic stem cell, or a        hematopoietic progenitor cell.    -   [16] The method of any one paragraphs 12-15, wherein the at        least one synthetic BCL11A microRNA is operably linked to a        promoter and constructed in a vector for expression in a        eukaryotic cell.    -   [17] The method of any one paragraphs 12-16, wherein the at        least one synthetic BCL11A microRNA is expressed from a RNA II        polymerase.    -   [18] The method of any one paragraphs 12-17, wherein the at        least one synthetic BCL11A microRNA is not expressed from a RNA        III polymerase.    -   [19] The method of paragraph 18, wherein the promoter is        selected from a group consisting of a spleen focus-forming virus        promoter, a tetracycline-inducible promoter, or a β-globin locus        control region and a β-globin promoter.    -   [20] The method of any one paragraphs 16-19, wherein the vector        is a virus.    -   [21] The method of paragraph 20, wherein the virus is a        lentivirus.    -   [22] The method of paragraph 21, wherein the lentivirus is        selected from the group consisting of: human immunodeficiency        virus type 1 (HIV-1), human immunodeficiency virus type 2        (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine        infectious anemia virus (EIAV), feline immunodeficiency virus        (FIV), bovine immune deficiency virus (BIV), and simian        immunodeficiency virus (SIV).    -   [23] An isolated nucleic acid molecule comprising the nucleotide        sequence selected from the group consisting of SEQ ID NOS: 1-10,        13-18, and 25-44.    -   [24] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 1.    -   [25] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 2.    -   [26] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 3.    -   [27] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 4.    -   [28] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 5.    -   [29] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 6.    -   [30] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 7.    -   [31] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 8.    -   [32] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 9.    -   [33] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 10.    -   [34] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 13.    -   [35] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 14.    -   [36] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 15.    -   [37] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 16.    -   [38] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 17.    -   [39] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 18.    -   [40] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 25.    -   [41] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 26.    -   [42] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 27.    -   [43] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 28.    -   [44] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 29.    -   [45] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 30.    -   [46] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 31.    -   [47] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 33.    -   [48] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 34.    -   [49] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 35.    -   [50] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 36.    -   [51] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 37.    -   [52] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 38.    -   [53] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 39.    -   [54] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 40.    -   [55] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO:41.    -   [56] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 42.    -   [57] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 43.    -   [58] The isolated nucleic acid molecule of paragraph 23, wherein        the molecule comprises the nucleotide sequence of SEQ ID NO: 44.    -   [59] A vector comprising the isolated nucleic acid molecule of        paragraph 23.    -   [60] The vector of paragraph 59, wherein the vector further        comprises a spleen focus-forming virus promoter, a        tetracycline-inducible promoter, or a β-globin locus control        region and a β-globin promoter.    -   [61] A host cell comprising the vector of paragraph 59 or 60.    -   [62] The cell of paragraph 61, wherein the cell is an embryonic        stem cell, a somatic stem cell, a progenitor cell, a bone marrow        cell, a hematopoietic stem cell, or a hematopoietic progenitor        cell.    -   [63] The cell of paragraph 61, wherein the cell is an        erythrocyte.    -   [64] A bacterium comprising the isolated nucleic acid molecule        of paragraph 23.    -   [65] A virus comprising the isolated nucleic acid molecule of        paragraph 23.    -   [66] The virus of paragraph 65, wherein the virus is a        lentivirus.    -   [67] The virus of paragraph 66, wherein the lentivirus is        selected from the group consisting of: human immunodeficiency        virus type 1 (HIV-1), human immunodeficiency virus type 2        (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine        infectious anemia virus (EIAV), feline immunodeficiency virus        (FIV), bovine immune deficiency virus (BIV), and simian        immunodeficiency virus (SIV).    -   [68] A composition comprising an isolated nucleic acid molecule        of any one of paragraphs 1-58, a vector of paragraphs 59 or 60,        a host cell of any one of paragraphs 61-63, or a virus of any        one of paragraphs 65-67.    -   [69] A composition comprising a vector of paragraphs 59 or 60, a        host cell of any one of paragraphs 61-63, or a virus of any one        of paragraphs 65-67.    -   [70] The composition of paragraph 68 or 69, further comprising a        pharmaceutically acceptable carrier or diluent.    -   [71] A composition of any one of paragraphs 68-70 for use in the        treatment or for reducing a risk of developing a        hemoglobinopathy in a subject.    -   [72] A composition of any one of paragraphs 68-70 for use in the        manufacture of medicament in treatment or for reducing a risk of        developing, a hemoglobinopathy in a subject.    -   [73] A composition of any one of paragraphs 68-70 for use in        increasing the fetal hemoglobin levels expressed by a cell.    -   [74] The composition of paragraph 73, wherein the cell is an        embryonic stem cell, a somatic stem cell, a progenitor cell, a        bone marrow cell, a hematopoietic stem cell, or a hematopoietic        progenitor cell.    -   [75] A method of treating, or reducing a risk of developing, a        hemoglobinopathy in a subject, the method comprising:        administering to the subject a therapeutically effective amount        of an isolated nucleic acid molecule of any one of paragraphs        1-58, a vector of paragraphs 59 or 60, a host cell of any one of        paragraphs 61-63, or a virus of any one of paragraphs 65-67 to        the subject, thereby treating, or reducing the risk of        developing, the hemoglobinopathy in the subject.    -   [76] A method of treating, or reducing a risk of developing, a        hemoglobinopathy in a subject, the method comprising:        administering to the subject a therapeutically effective amount        of a composition of any one of paragraphs 68-74 into the        subject, thereby treating, or reducing the risk of developing,        the hemoglobinopathy in the subject.    -   [77] A method of treating, or reducing a risk of developing a        hemoglobinopathy in a subject, the method comprising increasing        fetal hemoglobin levels expressed by a cell in the subject.    -   [78] The method of any one of paragraphs 75-77, the method        further comprising selecting a subject having a hemoglobinopathy        or is at risk of developing a hemoglobinopathy.    -   [79] The method of paragraph 78, wherein the hemoglobinopathy is        sickle cell disease or thalassemia.    -   [80] The method of any one of paragraphs 75-80, the method        further comprising administering to the subject a therapy        comprising oxygen, hydroxyurea, folic acid, or a blood        transfusion.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Materials and Methods

The typical PCR reaction conditions are as follows: 1× reaction buffer(consist of MgCl₂ at 1.5 mM; 3.0 mM; 4.5 mM (final concentration)); 0.2mM of each of dATP, dCTP, dGTP and dTTP; 25 pmol each primer; 50 ngtemplate DNA; 3-10% (v/v) DMSO to melt structure (this is optional) in atotal volume of 100 μl.

The following is the typical reaction conditions or setting on thermalcycler for the PCR reaction: 94° C. for 3-5 min, during this time add 1UDNA polymerase or set up reaction on ice and then put tubes into PCRmachine when it gets up to 94° C.; followed by 25 cycles of 94° C. for 1min; 60° C. for 1 min, and 70° C. for 1 min; and end with 4° C. till PCRsamples are used.

Example 1

Manufacturing Synthetic miRs

Three different synthetic miRs were constructed, two which target BCL11Aat different sites and a third non-targeting to act as a control. Eachof these miRs was inserted into a constitutive expressing vector, aTET-inducible vector, and an erythroid specific vector.

miRs are made by annealing complimentary oligonucleotides, which have 4base pair 5′ overlaps corresponding to the sticky end left by arestriction digest with BbsI.

BCL11A miR1 oligos: Sense (SEQ ID NO: 1)ACGCTCGCACAGAACACTCATGGATTaccatgtggtagagAATCCATGAG TGTTCTGTGCGAGAnti-sense (SEQ ID NO: 2)CGCACTCGCACAGAACACTCATGGATTactaccacatggagAATCCATGA GTGTTCTGTGCGABCL11A miR2 oligos: Sense (SEQ ID NO: 3)ACGCTCCAGAGGATGACGATTGTTTActccatgtggtagagTAAACAATC GTCATCCTCTGGagAnti-sense (SEQ ID NO: 4)CGCActCCAGAGGATGACGATTGTTTActctaccacatggagTAAACAAT CGTCATCCTCTGGaNon-targeting oligos: Sense (SEQ ID NO: 11)ACGCTCAACAAGATGAAGAGCACCAActccatgtggtagagTTGGTGCTC TTCATCTTGTTGAGAnti-sense (SEQ ID NO: 12)CGCACTCAACAAGATGAAGAGCACCAActctaccacatggagTTGGTGCT CTTCATCTTGTTGA

Oligonucleotide pairs were denatured and then re-annealed (as for oligocassette in LM-PCR protocol) following which the cassette was purifiedusing microcentrifuge concentration devices. In the meantime, plasmidO.6.pBKS(miR223) was digested with BbsI and purified by running out onan agarose gel (no treatment with alkaline phosphatase). Each oligocassette was then ligated into the digested O.6.pBKS construct andtransformed into competent bacteria (Stbl3). Bacterial clones werepicked and mini-prepped to prepare isolated vectors. The synthetic miRswere sequenced using primers miR223 SEQ FOR and miR223 SEQ REV usingDMSO to melt structure.

miR223 SEQ FOR (SEQ ID NO: 19) TAAGCTTGATATCGAATTCC miR223 SEQ REV(SEQ ID NO: 20) GCTCTAGAACTAGTGGATCC

Example 2 Manufacture of Constitutive miR Vectors

Each miR was cloned into the LeGO-V2 lentiviral backbone such thatVenus-miR expression is driven by the constitutive SFFV promoter.

Modification of the Venus cDNA. The Venus cDNA will be amplified via PCRto add a NaeI restriction site to the 5′ end (as well as maintain a goodKozak consensus sequence) and a NotI site to the 3′ end.

Venus NaeI FOR: (SEQ ID NO: 21) TTgccggcATGGTGAGCAAGGGCGAGGVenus NotI REV: (SEQ ID NO: 22) TAgcggccgcTTACTTGTACAGCTCGTCC

The PCR products were run out on an agarose gel and then purified. Thepurified PCR product were TA-cloned into vector PCR 2.1 TOPO(INVITROGEN™) using the TA cloning kit. Bacterial clones were picked andDNA mini-prepped. Using restriction digest analysis, clones wereselected that a) contain the Venus PCR product (EcoRI digest) and b)contain the clone in an orientation where the NotI that was added isnext to the NotI site in the polylinker (i.e., so that a NotI digestdoes not excise the PCR fragment, but instead just linearises thevector). These clones were then sequenced using M13Forward and Reverseprimers.

Insertion of the miR sequences into the Venus-PCR 2.1 TOPO plasmid. TheVenus -PCR 2.1 TOPO plasmid was digested with NotI, treated with calfintestinal alkaline phosphatase, then run out on an agarose gel andpurified. The synthetic miR constructs were excised from the 0.6.pBKSplasmid by double digest with NotI and PspOMI, following by purificationby agarose gel extraction. The digested miR inserts were ligated intothe Venus-PCR 2.1 TOPO plasmid and the ligation product was used totransform competent bacteria (Stbl3). Individual bacterial clones werepicked and mini-prepped. Plasmids that contain the miR insert in thecorrect orientation (i.e., yield the full fragment when digested withNotI and NaeI) were selected.

Insertion of the Venus-miR cassette into LeGO-V2. The Venus-miR cassettewas excised from PCR 2.1 TOPO by double digestion with NotI and NaeI,followed by treatment with Klenow large fragment to blunt the NotIoverhang. This cassette was purified by agarose gel extraction. LeGO-V2or LeGO G2 was digested with BamHI and EcoRI, which released theVenus/eGFP cDNA. This linearized vector was treated with Klenow largefragment to blunt the EcoRI and BamHI overhangs, followed bypurification of the vector by agarose gel electrophoresis. The purifiedVenus-miR cassette and the LeGO vector were ligated together, and theproduct was used to transform competent bacteria. Individual bacterialclones were picked and DNA mini-prepped. Clones that contain the insertin the correct orientation were selected and grown up and used in maxipreps to manufacture viral supernatant.

Example 3 Manufacture of Erythroid-Specific miR Vectors

A polyadenylation signal was attached to the Venus-miR cassettesmanufactured described above. The resulting Venus-miR-PolyA cassetteswere inserted in the anti-sense orientation into the erythroid specificpRRL-HS3-HS2-B-globin lentiviral vector provided by Guilianna Ferrari.

Modification of the BGH polyadenylation signal. The BGH polyA signal wasamplified via PCR to maintain the PspOMI restriction site at the 5′ endand add NaeI and NotI sites to the 3′ end.

BGHpA PspOMI FOR: (SEQ ID NO: 23) CGCTCGAGCATGCATCTAGAGGBGHpA NaeI/NotI REV: (SEQ ID NO: 24)TTgcggccgccggcCGCGCTTAATGCGCCGCTACAG

The PCR products were run out on an agarose gel and then purified. Thepurified PCR product was TA-cloned into vector PCR 2.1 TOPO(INVITROGEN™) using the TA cloning kit. Bacterial clones were picked andDNA mini-prepped. Using restriction digest analysis, clones wereselected that contain the BGHpA PCR product (EcoRI digest and/orNotI/PspOMI double digest). These clones were sequenced using M13Forwardand Reverse primers.

Insertion of the BGHpA sequence into the Venus-miR-PCR 2.1 TOPO plasmidsmanufactured described above. The BGHpA cassette was excised from PCR2.1 TOPO by digestion with PspOMI and NotI following which the insertwas purified by agarose gel extraction. The Venus-miR-PCR 2.1 TOPOconstructs manufactured in step B2 above were digested with NotI andsubsequently treated with calf intestinal alkaline phosphatase. Thelinearized Venus-miR-PCR 2.1 TOPO vector was purified by running out onan agarose gel. The BGHpA insert was ligated into the Venus-miR-PCR 2.1TOPO vector and the product used to transform competent bacteria(Stbl3). Individual bacterial clones will be picked and mini-prepped.Plasmids that contain the BGHpA sequence inserted in the correctorientation (yields the whole insert upon digestion with NaeI) wereselected.

Insertion of the Venus-miR-BGHpA cassette into pRRL-HS3-HS2-B-globinvector. The Venus-miR-BGHpA cassettes were excised from PCR 2.1 TOPO bydigestion with NaeI. These inserts were purified by agarose gelelectrophoresis. The pRRL-HS3-HS2-B-globin vector was digested withEcoRV and treated with calf intestinal alkaline phosphatase. Thelinearized vector was purified by agarose gel electrophoresis. TheVenus-miR-BGHpA cassettes were ligated into pRRL-HS3-HS2-B-globin andthe ligation product used to transform competent bacteria. Individualbacterial clones were picked and mini-prepped. Plasmids that contain theVenus-miR-BGHpA cassettes in the correct orientation in thepRRL-HS3-HS2-B-globin vector were grown up for maxi prep in order thatthey can be used to generate lentiviral supernatant.

Example 4 In Vitro Cell RNA Interference Experiments are Performed asFollows.

Murine erythroleukemia cells kept in culture in IMDM with FCS weretransduced on fibronectin with SFFV-LVs (NT=scrambled shRNA,miR-2=targeting shRNA) at MOI=2 and sorted for Venus fluorescence.Timepoint analyzed after transduction was day 7. Cells were >95% Venuspositive and 10⁶ cells were collected and RNA extracted, cDNA wasobtained by reverse transcription and real-time qPCR was performed forBCL11A and epsi-gamma globin mRNAs with Gapdh as an internal controltranscript (FIG. 3). A standard curve method was employed to quantifyexpression.

In Vivo RNA Interference Experiments in Mice are Performed as Follows.

BojJ donor derived LSK HSCs were transplanted into lethally irradiatedC57/BL6 mice after transduction on fibronectin with SFFV-LVs(NT=scrambled shRNA, miR-1=targeting shRNA) at MOI=2. Injected cell dosewas 100,000 cells per mouse. Venus positive WBC carrying animals at 4months were pooled (n=2) and bone marrow sorted for Venus fluorescenceafter viability stain (7-AAD) (FIG. 3). RNA extraction and qPCR wasperformed as above.

Example 5 LCR-LV

Murine erythroleukemia cells kept in culture in IMDM with FCS weretransduced on fibronectin with LCR-LVs (NT=scrambled shRNA,miR-1=targeting shRNA) at MOI=2 and MOI=100 and sorted for Venusfluorescence. Timepoint analyzed after transduction was day 7. Cellswere >95% Venus positive and 10⁶ cells were collected and RNA extracted,cDNA was obtained by reverse transcription and real-time qPCR wasperformed for BCL11A and epsi-gamma globin mRNAs with Gapdh as aninternal control transcript (FIG. 4). A standard curve method wasemployed to quantify expression.

TET-LV

Murine erythroleukemia cells kept in culture in IMDM with FCS weretransduced on fibronectin with TET-LVs (NT=scrambled shRNA,miR-1=targeting shRNA) at MOI=2 and sorted for Venus fluorescence afterexposure to doxycycline at differential concentrations. Timepointanalyzed after transduction was day 7. Cells were >95% Venus positiveand 10⁶ cells were collected and RNA extracted, cDNA was obtained byreverse transcription and real-time qPCR was performed for epsi-gammaglobin mRNA with Gapdh as an internal control transcript (FIG. 4). Astandard curve method was employed to quantify expression.

Example 6

Peripheral blood SCD-patient derived CD34+ circulating HSC werefractionated from discarded apheresis material (approximately 200 ml,10⁶ CD34+ cells). Cells were transduced with SFFV-LVs (NT=scrambledshRNA, miR-1=targeting shRNA) at MOI=2 on fibronectin and differentiatedas modified from Giarratana et al. (Nat Biotech 2005). Cells wereanalyzed maturational acquisition of erythroid surface markers (GPA,CD71) by flow cytometry. Erythroid cells sequentially acquireerythroblast and erythrocyte morphology and express Venus fluorescence.Cells are collected at terminal differentiation stage and RNA extractedand qPCR analysis performed to evaluate gamma-globin mRNA induction bymiR-1 SFFV-IV compared to scrambled (NT) control (FIG. 5).

Example 7

Peripheral blood SCD-patient derived CD34+ circulating HSC werefractionated from discarded apheresis material (approximately 200 ml,10⁶ CD34+ cells). Cells were transduced with LCR-LVs (NT=scrambledshRNA, miR-1=targeting shRNA) at MOI=2 on fibronectin and injected at30,000 cells/animal into sub-lethally irradiated NSG mice without priorsorting. Animals were bled at 4 weeks post-injection and RBCs fixed andpermeabilized. HbF stain was performed and identified a LCR-LV-miR-1animal with human HbF levels at 10% (FIG. 6).

Example 8

Cord blood derived CD34+ human HSCs were transduced on fibronectin withSFFV-LVs (NT=scrambled shRNA, miR-1=targeting shRNA) at MOI=2 and sortedfor Venus fluorescence. Cells were also visualized by fluorescentmicroscopy on MS-5 stroma. The cells were differentiated along theB-lymphopoietic path by methods modified from Luo et al. (Blood 2009).Cells were analyzed weekly for the acquisition of mature B-lymphocytesurface markers and loss of immature progenitor markers to identify ablock in differentiation caused by the knock-down of BCL11A viaSFFV-LVs. Cells were collected at weekly timepoints, and RNA extractedto verify BCL11A mRNA knockdown by shRNA targeting via SFFV-LV-miR-1(FIG. 7).

Example 9

Optimization of Lentivirus Vector RNA Polymerase H Driven microRNAEmbedded shRNAs for Enhanced Processing and Efficient Knockdown ofBCL11A for Induction of Fetal Hemoglobin in Erythroid Cells.

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs)expressed via pol III promoters has been widely exploited to modulategene expression in a variety of mammalian cell types. To achievelineage-specific targeting of mRNAs, expression of shRNAs via pol IIpromoters is required, necessitating embedding the shRNA in mammalianmicroRNA (shRNAmiR) sequences for expression and processing. Here, inorder to achieve knockdown of the BCL11A transcription factor inhematopoietic cells, which has direct translational application inhemoglobinopathies, we compared the efficiency of mRNA modulation viapol III vs pol II based lentiviral vectors. Repression of the BCL11Aprotein could represent a therapeutic target for sickle cell disease andβ-thalassemias, as its knock-down has been shown to induce theexpression of the fetal HBG (γ-globin) gene ultimately leading toenhance levels of the fetal hemoglobin tetramer (HbF, α2γ2). In themouse, BCL11A is a key repressor of murine Hbb-y gene representing amurine HBG homolog. The inventors demonstrate up to 100-1000 fold lowerHbb-y induction due to reduced BCL11A knockdown efficiency usingshRNAmiR vs pol III mediated shRNA vector backbones. In order tounderstand the molecular basis for these differences, the inventorsconducted small RNA sequence analysis of cells transduced by multipleshRNA-shRNAmiR pairs. The inventors show that shRNAs expressed via a U6pol III promoter yield guide strand sequences that differ by a 4 bpshift compared to pol 11 driven (shRNAmiR) mature guide strandsequences. RNA sequencing demonstrated that the stretch of uridinesmaking up part of the pol III termination signal is transcribed andincluded at the 3′ end of the mature shRNA in a pol III vector backbone.The absence of these additional sequences is associated with acorresponding shift in the dicer cleavage site, thereby generating adifferent mature shRNA with an alternate seed sequence influencing theefficacy of target gene knockdown in pol II based vectors. In addition,both the absolute abundance and the ratio of guide to passenger strandare significantly different in cells transduced with either pol II orpol III based vectors. Incorporating a 4 bp shift in the guide strand ofshRNAmiR resulted in a faithfully processed (a mature guide strandsequence identical to U6-driven sh-RNAs) shRNA sequence and improvedknock-down efficiency of BCL11A by 50-70% at the protein level and wasassociated with a 100-300-fold enhancement of Hbb-y induction in murineerythroleukemia cells. The inventors have discovered a modified strategyfor the prospective design of shRNAmiR vector backbones to achievelineage-specific regulation of target genes.

Example 10

Optimization of miRNA-Embedded shRNAs for Lineage-Specific BCL11AKnockdown and Hemoglobin F Induction.

Materials and Methods

Design and Screening of shRNAs

U6 promoter-driven lentiviral vectors (pol III-puro) expressingdifferent shRNAs targeting BCL11A BCL11A mRNA were obtained from theBroad Institute (Cambridge, Mass.). The pol III-puro has hPGK promoterdriven puromycin selection marker. More than 100 shRNAs targeting eitherboth XL/L forms or only XL form and 3′UTR region were screened in MELcells in a 96 well format using a Qiagen Turbocapture plate and with amultiplexed Taqman qRT-PCR reaction measuring Gapdh and Hbb-y.

Construction of shRNAmiR Constructs

The shRNAmiR, vectors were constructed by cloning the shRNA sequenceswith flanking mir223 sequences into the lentiviral LeGO-V2 vectorcontaining a SFFV-driven Venus reporter (28). The shRNAmiR sequenceswith mir223 loop were synthesized by genscript USA Inc. (NJ, USA) andthe resulting shRNA^(miR)S were cloned into the pol II backbonedownstream of the Venus coding sequence using Xba1 and BamH1 sites. Allthe sequences of shRNAs are listed in FIG. 21A. A non-targeting controlshRNA sequence was designed and named as SFFV-shRNAmiRNT or NT in shortform. The SFFV-GFP vector, not containing any shRNA cassette andexpressing GFP via an SFFV promoter, was used as a mock control (33).

Virus Production and Titration

Lentiviral vector supernatants were generated by co-transfecting 10 μgof lentiviral transfer vectors, 10 μg of gag-pol, 5 μg of rev and 2 μgof VSVG packaging plasmids into HEK293T cells in a 10 cm culture dishusing calcium phosphate reagent (INVITROGEN™) Supernatants werecollected at 24 h and 48 h after transfection, filtered through a 0.4micron membrane (CORNING®, NY, USA) and subsequently concentrated byultracentrifugation at 23000 rpm for 2 h in a Beckmann XL-90 centrifugeusing SW-28 swinging buckets. To determine the titer, NIH3T3 cells wereinfected with the virus in the presence of polybrene (8 μg/ml) andanalyzed 48 h post-transduction by FACS for Venus expression (pol IIconstructs) or by puromycin selection (1 mg/ml, pol III constructs).

Cell Culture

3T3, 293T and MEL cells were maintained in DULBECCO's modified Eagle'sor RPMI medium supplemented with 10% fetal calf serum, 2%penicillin-streptomycin and 2 mM glutamine, respectively.

In Vitro Erythroid Differentiation Culture

Frozen stocks of primary human CD34+ cells were obtained from mobilizedperipheral blood of healthy donors (Center of Excellence in MolecularHematology at Fred Hutchinson Cancer Research Center, Seattle or theFlow Core at Boston Children's Hospital) according a protocol approvedby the BCH Institutional Review Board. Erythroid differentiationprotocol used is based on a 3-phase protocol adapted from (48). Thecells were cultured in erythroid differentiation medium (EDM) based onIMDM (Iscove modified DULBECCO's medium), (CELLGRO®) supplemented withstabilized glutamine, 330 μg/mL holo-human transferrin (SIGMA®), 10μg/mL recombinant human insulin (SIGMA®), 2 IU/mL heparin Choay (SIGMA®)and 5% solvent/detergent virus inactivated (S/D) plasma. During thefirst phase of expansion (days 0 to 7), CD34+ cells were cultured in EDM(erythroid differentiated medium) in the presence of 10-6 Mhydrocortisone (HC) (SIGMA®), 100 ng/mL SCF (R&D SYSTEMS™), 5 ng/mL IL-3(R&D SYSTEMS™), ) and 3 IU/mL Epo (AMGEN®). On day 4, cells wereresuspended in EDM containing SCF, IL-3, Epo and HC. In the second phase(days 7 to 11), the cells were resuspended in EDM supplemented with SCFand Epo. In the third phase (day 11 to day 18), the cells were culturedin EDM supplemented with Epo alone. The cultures were maintained at 37°C. in 5% CO₂ in air.

Transduction and Flow Cytometry for In Vitro Culture

MEL and CD34+ cells were transduced with lentiviral vectors expressingU6-shRNA or SFFV-shRNAmiR in the presence of polybrene (8 μg/ml)(SIGMA-ALDRICH® Corp. St. Louis, Mo., US) for MEL cells and 10 μMprostaglandin E2 and 2 μg/ml polybrene for CD34+ cells rand centrifugedfor two hours at (2000 rpm) at room temperature. Live cells were eithersorted for Venus expression (pol II vectors) 48h post transduction byusing BD FACS Aria II (BD BIOSCIENCES®) or cells were selected in thepresence of puromycin (1 mg/ml, pol III constructs). For FACS analysis7AAD (INVITROGEN™) was included as dead cell marker. CD34+ cells werelabeled with Allophycocyanin (APC), phycoerythrin (PE), and PE-Cyanine7conjugated antibodies. Anti-CD235 (glycophorin A) -PE, anti-CD71-APC, oranti-CD71-PE-Cyanine7 antibodies and DRAQ-5 (all EBIOSCIENCE®) were usedfor phenotyping. Analyses were performed on LSR-II flow cytometer(BECTON DICKINSON®) using Diva or FloJoX (TREESTAR™) software.Isolation, transduction and flow cytometry for mouse transplantationexperiments

Lineage negative mouse bone marrow cells were isolated by flushingfemur, tibia and hip of CD45.1 BoyJ (B6.SJL-Ptprca Pepcb/BoyJ) andCD45.2 B6 mice (C57BL/6J) followed by lineage depletion using the MouseLineage Cell depletion kit (Miltenyi, Biotec Inc., San Diego, USA).Cells were cultured at a density of 0.2-1×10⁶ cells/ml in 100 ng/mlmSCF, 20 ng/ml mIL-3 (both PEPROTECH®, Rocky Hill, USA), 100 ng/mlhFlt3-L and 100 ng/ml hTPO (both CELLGENIX®, Portsmouth, USA) inSTEMSPAN™ SFEM medium (STEMCELL® Technologies, Vancouver, CA). Following24 h pre-stimulation cells were transduced at a density of 1×10⁶cells/ml at an MOI of 40 and transplanted into lethally irradiated(7+4Gy, split dose) recipients three days after isolation. Forcompetitive repopulation experiments, equal numbers of cells fromdifferent transduction groups were mixed prior to transplantation intoCD45.2 or heterozygous CD45.1/CD45.2 double positive recipients(0.4-1×10⁶ per animal). Cell mixtures were analyzed via flow cytometryto confirm equal contributions of both competitor cell fractions, andreadjusted if required. Analysis of peripheral blood, bone marrow andspleen was performed at multiple time points using the followingantibodies: CD45.1, CD45.2, B220, CD11b, CD3, CD71, Ter119 and fixiableviability dye EFLUOR780@. For analysis of the erythroid lineage redblood cell lysis was omitted. Analyses were performed on LSR-II orLSRFortessa flow cytometers (BECTON DICKINSON®) and Diva or FloJoX(Treestar™) software. Data analyses and statistics were done using Excel(MICROSOFT®) and GRAPHPAD PRISM® 5.

For transplantation of hCD34 cells ˜10 week old female NSG-mice(NOD/LtSz-scid Il2rg−/−) (Jackson Laboratory, Bar Harbor, Me.) wereirradiated with 2.7Gy followed by injection of ˜10⁶ cells per animalthree days post isolation. Irradiated mice were fed BAYTRIL®supplemented water for 14 days.

RNA Extraction and qRT-PCR

Total RNA was extracted from MEL cells 7 days after sorting/postselection with puromycin, or freshly sorted cells on day 18 of erythroiddifferentiation of CD34+ cells, using the QIAGEN® RNA Plus micro kit(Valencia, Calif.). CDNA was generated using random hexamer primers andsuperscript III (INVITROGEN™, Carlsbad, Calif.). Quantitative PCR wasperformed using SYBR® Green PCR master mix (APPLIED BIOSYSTEMS®,Warrington UK) with intron spanning mouse Hbb-y and Gapdh primers (Hbb-yforward 5′-TGGCCTGTGGAGTAAGGTCAA-3′ (SEQ ID NO: 98), reverse5′-GAAGCAGAGGACAAGTTCCCA-3′(SEQ. ID. NO:69)), (Gapdh forward5′-TCACCACCATGGAGAAGGC-3′ (SEQ. ID. NO:70), reverse5′-GCTAAGCAGTTGGTGGTGCA-3′ (SEQ. ID. NO:71)) and human HBG, HBB andGAPDH primers (HBG forward 5′-TGGATGATCTCAAGGGCAC-3′ (SEQ. ID. NO:72),reverse 5′-TCAGTGGTATCTGGAGGACA-3′ (SEQ. ID. NO:73)) (HBB forward5′-CTGAGGAGAAGTCTGCCGTTA-3′ (SEQ. ID. NO:74), reverse5′-AGCATCAGGAGTGGACAGAT-3′ (SEQ. ID. NO:75)) and GAPDH forward5′-ACCCAGAAGACTGTGGATGG-3′ (SEQ. ID. NO:76), reverse5′-TTCAGCTCAGGGATGACCTT-3′ (SEQ. ID. NO:77)). The PCR amplificationconditions were: 95° C. for 10 min, followed by 40 cycles of 15 see at95° C. and 1 min at 60° C. The qPCRs were performed on aABI® 7500machine (APPLIED BIOSYSTEMS®, Foster City, Calif.). A standard curveusing serial dilutions of cDNAs was used to determine the preciseamplification efficacy for each reaction. Hbb-y and γ-globin expressionlevels were normalized to GAPDH as an internal control, and relativegene expression (ΔΔCt method) was used for analysis of PCR data,including correction for differential amplification efficiencies.

Northern Blot Analysis

MEL cells transduced with U6-shRNAs and SFFV-shRNA^(miR)s were sortedand collected after puromycin selection culturing for 7 days. Total RNAwas isolated using 1 ml TRIZOL® reagent (AMBION®), and 15 μg wereresolved on a 15% acrylamide gel. Small transcript sizes were determinedusing the Decade Ladder (AMBION®, Austin, Tex.). RNA was transferred toHYBOND™-XL membrane (AMERSHAM™, Piscataway, N.J.) and UV-crosslinked.Blots were pre-hybridized using UltraHyb-Oligo (AMBION®, Austin, Tx) at35° C., probed with γ-32P-labeled oligonucleotides (4 polynucleotidekinase; AMERSHAM™, Piscataway, N.J.) at 37° C. for one hour, washed in2× sodium citrate, 0.1% sodium dodecyl sulphate at 30-35° C., andexposed to film. Probe sequences for detecting mature miRNA were asfollows:

(SEQ. ID. NO: 78) shRNA1, 5′ CGGAGACTCCAGACAATCGC 3′; (SEQ. ID. NO: 79)shRNA2, 5′ CTCCAGGCAGCTCAAAGATC 3′; (SEQ. ID. NO: 80)shRNA3, 5′ TCTCTTGCAACACGCACAGA 3′; (SEQ. ID. NO: 81)shRNA4, 5′ CAGGACTAGGTGCAGAATGT 3′; (SEQ. ID. NO: 82)shRNA5, 5′ ATCGAGTGTTGAATAATGAT 3′; (SEQ. ID. NO: 83)shRNA6, 5′ GTACCCTGGAGAAACACAT 3′; (SEQ. ID. NO: 84)shRNA7, 5′ ACTGTCCACAGGAGAAGCCA 3′; (SEQ. ID. NO: 85)shRNA8, 5′ CAGTACCCTGGAGAAACACA 3′.

Western Blot Analysis

Transduced MELs and CD34+ cells were lysed in lysis buffer (RIPA) withprotease (ROCHE®) and phosphatase inhibitors (SANTA CRUZ BIOTECHNOLGY®),pepstatin and leupeptin (SIGMA). Protein lysates were estimated by BCAprotein assay (THERMO SCIENTIFIC). 25 μg of protein was suspended in 2×Laemmli sample buffer, boiled and loaded on to a 8% SDS-poly-acrylamidegel and subsequently transferred to a Polyvinylidene fluoride (PVDF)membrane (MILLIPORE®). Following blocking in PBS with 0.1% Triton-X100and 5% nonfat dry milk, the PVDF membrane was incubated with amonoclonal mouse anti-BCL11A antibody (ABCAM®) or mouse anti-β-actin(SIGMA®). Anti-mouse IgG HRP-linked secondary antibody (CELL SIGNALING®)was used for detection by chemiluminescence 20× LUMIGLO® Reagent and 20×Peroxide (CELL SIGNALING®).

HPLC Analysis

Hemolysates were prepared from cells on day 18 of differentiation usingosmotic lysis in water and three rapid freeze-thaw cycles. Hemoglobinelectrophoresis with cellulose acetate and high performance liquidchromatography (HPLC) were carried out with the lysates, in the clinicallaboratories of the Brigham and Women's Hospital usingclinically-calibrated standards for the human hemoglobins.

RNA Sequencing and Analysis

Small RNAs were extracted from 6×10⁶ MEL cells using mirVana miRNAisolation kit (INVITROGEN™) according to the manufacturer's instructionsand sent out for deep RNA sequencing using ILLUMINA® Hiseq2000. Aself-developed PERL script was used to remove the adaptor sequence, and19-25nt small RNAs were used for further analysis. The BOWTIE software(obtained from the internet website at bowtie-bio period sourceforgeperiod net) was used for alignment, and 1 mismatch was permitted.Expression level of small RNAs was normalized to one million of totalreads of each library for comparison between different samples. For theexperiment with 250 TRC shRNAs in 4 cell lines, lentivirus was preparedby the Broad Institute using a high-throughput virus preparationprotocol and cells were infected at high MOI with a single shRNA perwell in 96 well plates (the protocols are obtained from the internetwebsite of the Broad Institute at Cambridge, Mass., USA, at the RNAipublic resources section under “puromycin”) was added at 1 daypost-infection and cells were lysed in TRIZOL® at 4 days post-infection.All lysates were pooled for each cell line, followed by total RNAextraction and small RNA library preparation (49). ILLUMINA® reads weretrimmed, collapsed to unique reads (>17nt) with counts, and mapped toTRC shRNA expression vector sequences allowing no mismatches. MatureshRNA sequence distributions were calculated for each shRNA beforeaveraging across shRNAs.

Statistical Analysis

The GRAPHPAD PRISM® 5.0 software package was used for statisticalanalysis. Results are expressed as mean±standard deviation (SD).Statistical significance was assessed by t-test.

Results

Decreased Knockdown Efficiency of BCL11A by shRNAs Embedded in amicroRNA Scaffold (shRNAmiR) Compared to Simple Stem-Loop shRNAs

To identify candidate shRNAs mediating effective knockdown of BCL11A, alentiviral library of 118 shRNAs targeting coding sequences of BCL11AmRNA conserved between humans and mice was screened in MEL cells. ShRNAswere expressed from a pol III based U6 promoter (FIG. 19A, left panel)in the LKO lentivirus backbone (26) containing a puromycin resistancegene for selection named LKO-U6-BCL11A-shRNA^(miR) (hereafter U6-shRNA).MEL cells, a commonly used cell line for the study of globin generegulation, were transduced with the lentivirus vectors expressingshRNAs at a multiplicity of infection (MOI) of 2. The normalizedexpression of embryonic mouse Hbb-y mRNA, which serves as a functionalhomolog of the human γ-globin gene (27) provides an indirect readout ofBCL11A knockdown (FIG. 19B, y-axis). As a second readout, the shRNA poolwas also screened using a MEL-reporter cell line harboring a mCherryknock-in at the Hbb-y locus (D. Bauer, unpublished). Fluorescentreporter induction was analyzed by flow cytometry (FIG. 19B, x-axis).Eight shRNAs (labeled and named as shRNA1 through 8 in FIG. 19B) thatconsistently induced Hbb-y and mCherry reporter expression in MEL cellswere cloned into human microRNA223 (miR-223) flanking and loop sequencesto create synthetic microRNAs (shRNAmiR) with the goal of developinglineage-specific expression vectors for knockdown of BCL11A. For initialanalysis, this cassette was incorporated in the pLeGO lentiviral vector(28) (FIG. 19A, right panel) into the 3′ untranslated region of theVenus fluorescent reporter under control of the strong and ubiquitouslyexpressed spleen focus forming virus (SFFV) promoter/enhancer namedLEGO-SFFV-BCL11A-shRNAmiR (hereafter SFFV-shRNAmiR).

The knockdown efficacy of shRNAs that incorporated the same 21-basetarget-matching sequences was directly compared, but in the context ofthe pol III and pol II expression cassettes (i.e. U6-shRNAs vsSFFV-shRNAmiRs.) in MEL cells using a non-targeting (NT) shRNA asnegative control. BCL11A protein was detected by immunoblot in celllysates from MEL-cells transduced at an MOI of 2 (FIG. 19C). Knockdownof BCL11A was consistently less efficient in cells expressingSFFV-shRNAmiR compared to U6-shRNAs (FIG. 19C). To confirm thefunctional significance of this difference, the inventors measuredinduction of Hbb-y mRNA levels by qRT-PCR (FIG. 19D) in homogeneouspopulations of transduced cells obtained either by puromycin selectionor by fluorescence-activated cell sorting (FACS). The reduced knockdownefficiency of SFFV-shRNAmiRs as compared to U6-shRNAs (see FIG. 19C)translated into significantly less induction of Hbb-y by SFFV-shRNAmiRs(FIG. 19D) and the differences in Hbb-y induction appear more pronouncedthan the differences in BCL11A knockdown.

SFFV-shRNAmiR and U6-shRNAs Give Rise to Different Mature Guide StrandSequences

To understand the molecular basis for these differences, sequencing ofsmall RNAs from cells transduced with various U6-shRNAs were performedand their corresponding SFFV-shRNAmiR counterparts. It was hypothesizedthat significant differences in Hbb-y induction were due to theproduction of different mature guide strands from the distinctshRNA-containing transcripts that are produced from the pol II and polIII contexts. The processed guide strand sequences from both theU6-shRNAs and SFFV-shRNAmiR contexts were therefore assessed (FIGS. 20Aand B). The most abundantly found mature guide RNAs produced fromSFFV-shRNAmiRs closely corresponded to the in silico predicted maturesequence (FIG. 20B). In contrast, most of the U6-shRNAs yielded matureguide strand sequences that match the predicted Dicer product consistingof -22nt of the 3′ end of the pol III transcript, including a stretch of3-5 nt derived from the pol III termination signal, but lacking acorresponding number of nucleotides of the target matched sequence atthe 5′ end. A similar distribution of processed products were observedin a large scale screen of 247 different U6-shRNAs in A549, MCF7, Jurkatand U937 cell lines, in which the predominant guide strand sequence hasan average length of 22nt with its 5′ end starting 4 bases from theconstant loop sequence. Deep sequencing of 247 processed TRC shRNAproducts in these four cell lines were performed (FIG. 25). The resultsindicate that the predominant mature guide strand starts at position 4of the antisense sequence of the shRNA and includes four 3′-terminal Uresidues. Processing was generally consistent among cell lines and amongdifferent shRNA sequences. The average read frequency for each maturesequence is weighted equally across shRNAs, although some shRNAsgenerated >1,000-fold more reads than others. The semiquantitativenature of small RNA sequencing, due to strong RNA ligase biases duringlibrary preparation, make comparisons of relative expression orprocessing levels impossible, but consistent trends across cell linesand shRNAs demonstrate the likely predominant guide strand identity.Sense strand reads were also detected (<30% per shRNA on average), withthe vast majority starting with ‘GG’ and extending 20nt into the sensestrand sequence. These mature sequences are exactly consistent with aDicer product of the primary hU6 shRNA transcript, with no need toinvoke a Drosha/DCGR8 processing step. Taken together with, thesefindings indicate the importance of considering the processing eventsthat generate mature sequences from pol II shRNAmiR and pol III shRNAtranscripts when transferring shRNA sequences between vectors. The verysimilar distributions of mature sequences observed for the four celltypes that were studied suggests that these processing patterns willgeneralize across different cellular contexts. The differences in themature guide strand sequence generated in pol III vs pol II basedvectors contribute substantially to differential BCL11A down regulationobserved with U6-shRNAs compared to SFFV-shRNAmiRs. These data suggestpredicted conversions between pol III and pol II vectors may be possibleby considering the Drosha and Dicer cleavage of pol II shRNAs comparedto the Dicer cleavage of pol III shRNAs.

Modification of shRNA Sequences in a Pol H Based Vector Leads toImproved Knockdown Efficiency

Based on these findings, the inventors hypothesized that using thepredicted mature sequence from pol III shRNA vectors when transferringsequences into SFFV-shRNAmiR would lead to enhanced knockdownefficiency. Therefore, a set of SFFV-shRNAmiRs containing a 4-nucleotideshift in the 5′ end of the guide strand sequence were designed (FIG.21A). At the 3′-end the nucleotides GCGC were added to achieve higher3′-end thermodynamic stability in the siRNA duplex which should promotepreferential RISC-loading of the intended guide strand. The effect ofmodifications on knockdown efficiency and Hbb-y induction was evaluatedin MEL cells by immunoblot and qRT-PCR, respectively. Improved knockdownefficiency of BCL11A protein was observed with SFFV-shRNAmiR1, 3 and 8(FIG. 21B). The enhanced knockdown correlated with a 200 to 400 foldincreased induction of Hbb-y transcripts (FIG. 21C). The otherSFFV-shRNAmiRs did not show an appreciable increase in knockdownefficiency. To understand more fully the mechanism underlying theimproved efficiency of modified SFFV-shRNAmiRs 1, 3 and 8 were analyzedthe abundance of guide and passenger strand small RNAs and their ratiosby Northern blot. First, a higher abundance of guide strand was seen forpol III versus pol II vectors in all cases. Furthermore, particularlyfor modified SFFV-shRNAmiRs 1 and 3 a higher abundance and higher guideto passenger strand ratios were found versus the unmodified shRNAmiRs,while these parameters were not affected for SFFV-shRNA8 (FIG. 21D).Deep sequencing of small RNAs was performed to evaluate the impact ofthe modification on guide strand sequences and to correlate it with thechanges observed in BCL11A knockdown. Generally, the resulting processedsequences reflected the introduced 4 nt shift, resulting in a guidestrand with seed regions similar to the sequences obtained from pol IIIshRNAs expressed in the LKO backbone. For SFFV-shRNAmiRs 1, 3 and 8, asingle dominant sequence was found, which contrasts the less effectiveSFFV-shRNAmiRs which showed a broader distribution of sequences.

Effect of shRNAmiR Modification on BCL11A Knockdown and γ-GlobinInduction in Primary Human CD34+ Derived Erythroid Cells

Reactivation of fetal globin with BCL11A knockdown has therapeuticpotential for the treatment of sickle cell disease and β-thalassemia. Toevaluate the effect of modified SFFV-shRNAmiR on knockdown efficiency ofBCL11A and induction of γ-globin and HbF expression in primary humancells, G-CSF mobilized peripheral blood (mPB) CD34+ HSPCs from healthyvolunteers were transduced with vectors expressing U6-shRNAs,SFFV-shRNAmiR and modified SFFV-shRNAmiR and then subjected to erythroiddifferentiation. After eleven days in culture, BCL11A levels weredetermined via western blot (FIG. 22A). Consistent with findings in MELcells, enhanced knockdown was observed with modified SFFV-shRNAmiRs 1,3, and 8, which also led to increased induction of γ-globin transcripts(FIG. 22B). The status of erythroid differentiation was assessed at day18 of culture by flow cytometric analysis for surface expression of CD71and GpA and enucleation. No significant differences were observedbetween SFFV-shRNAmiRs and control vector transduced samples (FIG. 22C).In contrast, U6-shRNAs led to mild delay in the acquisition ofdifferentiation markers during the later phases of maturation, whichcould indicate toxicity due to U6-promoter mediated shRNAoverexpression. The observations of high γ-globin mRNA induction wereconfirmed by increased HbF protein measured by high performance liquidchromatography (HPLC). All three tested modified shRNAmiRs yieldedincreased HbF output compared to unmodified SFFV-shRNAmiRs (FIG. 22D),where between 40-50% of total hemoglobin in the erythroid cells was HbF.The correlation between γ-globin mRNA and HbF protein was high(r2=0.96), supporting the reliability of the analyses (FIG. 22E).

In summary, the inventors have demonstrated that shRNAs embedded into amiRNA scaffold and expressed via pol II promoters are processed to yielddiffering mature siRNAs in transduced cells compared with siRNAexpressed from the U6 promoter. The target-matched sequence in themature shRNA derived from the pol III promoter construct is uniformlyshifted 3′ by 3-5 nt and this difference was associated with significantdifferences in knockdown efficiency of the target transcript. In thecase of BCL11A, a potential therapeutic target, this led to appreciabledifferences in the reactivation of γ-globin expression. These datademonstrate the importance of design optimization when transferringshRNA sequences into a microRNA scaffold to allow for pol II mediatedexpression.

Ubiquitous Knockdown of BCL11A in Hematopoietic Stem and ProgenitorCells (HSPCs) Impairs Hematopoietic Reconstitution and can beCircumvented by Targeting shRNAmiR Expression to Erythroid Cells

The impact of SFFV-shRNAmiR expression in vivo was assessed in a mousemodel of HSPC transduction and transplantation. Lineage-negative (lin−)HSPCs from the bone marrow of β-YAC mice expressing the CD45.2 cellsurface maker were transduced ex vivo with SFFV-shRNAmiR vectors or anon-targeting vector (SFFV-shRNAmiRNT) and transplanted into lethallyirradiated CD45.1 BoyJ-recipient mice. Untransduced cells weretransplanted in a control group. β-YAC mice harbor the human β-globinlocus as a transgene that is developmentally regulated in the mouseenvironment, showing differential expression of fetal and adult β-globingenes. For validation purposes and for better comparison with previouslypublished data a well described shRNA (23, 27, 29) (here termedshRNAmiR*) embedded into miRNA223 flanking sequences was employed. At 4,8 and 12 weeks after transplantation, donor cell engraftment wasdetermined based on CD45.1 and CD45.2 chimerism (FIG. 23A). Donor cellengraftment followed the expected pattern with near complete engraftmentin peripheral blood (PB) and bone marrow (BM) after 8 weeks. However,unexpectedly, the fraction of gene modified cells steeply declined overtime (FIG. 23B). Despite initial transduction rates of ˜40% using theBCL11A knockdown vector, gene marking at week 12 was only 2-3% of totaldonor derived CD45 cells. Overexpression of the SFFV-shRNAmiR NT wasalso associated with reduced engraftment of gene modified cells but to alesser extent, indicating both sequence-specific and non-specifictoxicity in the engrafting HSPC cells. The timing of the loss of donorcells expressing shRNAs suggests an effect on the more primitivehematopoietic stem cell compartment.

To further investigate the negative impact on hematopoieticreconstitution, quantitative competitive repopulation experiments wereperformed (FIG. 23C-23F). Lineage negative cells from CD45.1 (BoyJ) andCD45.2 (Bl\6) donor animals were transduced with various vectorsexpressing SFFV-shRNAmiRs against BCL11A, a shRNA^(miR)NT or only a bluefluorescent protein (BFP) reporter under control of the ubiquitouslyexpressed SFFV-promoter (SFFV-BFP). Cells were transplanted intocongenic CD45.1/CD45.2 animals, allowing for identification of bothdonor populations and the recipient cells. In experiments in which theSFFV-BFP vector was employed, CD45.1 donor cells were transplanted intoCD45.2 animals and the transduced donor cell populations were identifiedand compared based on fluorescence. Prior to transplantation, equalnumbers of cells of the two populations transduced with competingvectors were mixed. The final ratio of gene modified cells obtained withboth vectors in the transplanted population was analyzed via flowcytometry which confirmed comparable transduction rates ranging from55-70% (FIG. 23C). The contribution of gene modified cells was assessedin transplanted animals in peripheral blood, bone marrow and spleen 4, 8and 12 weeks after transplantation (FIG. 23D) and minor differences inthe ratio of the infused transduced cells were taken into account forthis analysis. In all instances and at each time point, cells transducedwith vectors targeting BCL11A were outcompeted by cells transduced withthe NT or SFFV-BFP vector, indicating a selective disadvantage uponBCL11A knockdown. No significant differences in reconstitution ofhematopoietic cells compared to the ratio of the initially transplantedpopulation was observed when two BCL11A targeting vectors competedagainst each other. Consistent with the findings in FIG. 23B, theoverexpression of shRNAmiRNT also had a negative impact on hematopoieticreconstitution, as this group was outcompeted by cells transduced by avector expressing only SFFV-BFP and not expressing a shRNA. Theinventors performed a more detailed analysis of the B lymphocyte andmore primitive HSC compartment within the transduced fraction of bonemarrow cells (FIG. 23E). As anticipated from previous studies showing anabsence of B cells in BCL11A−/− mice (30, 31) the number of B220positive B-cells was significantly reduced upon BCL11A knockdown.Although not reaching significance, there was a trend toward loss ofmore primitive lin−, Sca-1+, c-kit+(LSK) cells that include theengrafting HSC compartment.

Erythroid specific knockdown of BCL11A could potentially circumvent theadverse effect of BCL11A knockdown on HSCs and B cells, whilemaintaining the therapeutic effect of γ-globin induction in erythroidcells. To direct knockdown selectively to erythroid cells, a lentivirusvector was generated in which the shRNAmiR cassette and the Venusfluorescent reporter is expressed under the control of the minimalβ-globin proximal promoter linked to hypersensitive sites 2 and 3 (HS2and HS3) of the β-globin locus control region (LCR) (32) (FIG. 23F)named LV-LCR-BCL11A-shRNAmiR (hereafter LCR-shRNAmiR). The expressionprofile of the Venus reporter transgene in the engrafted hematopoieticcell populations in vivo was first assessed in mice transplanted withtransduced HSPC (FIG. 23G and FIG. 26). In FIG. 26, lineage negativecells were transduced using the LCR-shRNAmiR vector and engrafted intolethally irradiated recipient mice. Twelve weeks later donor cells anddifferent hematopoietic cell types were identified using surfacemarkers. Shown here is a representative gating scheme and histogramblots showing Venus expression in various lineages. Numbers in blotsindicate the percentage of venus positive cells and mean fluorescenceintensities (MFI). Expression of the transgene was tightly regulated;with no detectable expression in LSK and B cell fractions, very lowlevels of expression in T-cells and low levels of expression in myeloidcells in some animals. In contrast, transgene expression was stronglyupregulated during erythroid differentiation, beginning in CD71+/Ter119−cells representing erythroid progenitors and pro-erythroblasts andpeaking in the CD71+/Ter119+ double positive stage, representingbasophilic erythroblasts. During final stages of erythroid maturation, alarge fraction of CD71−/Ter119+ cells representing reticulocytes andmature erythrocytes expressed the reporter at a similar percentagecompared to CD71+/Ter119+ cells.

Next, to determine whether use of the LCR-vector circumvents thereconstitution defect observed upon ubiquitous SFFV-shRNAmiRoverexpression, a competitive transplantation experiment was performedusing LCR-shRNAmiR and SFFV-shRNAmiR (FIG. 23H). As the LCR-vector istranscriptionally silent in lin− cells, an aliquot of cells to be usedfor transplantation was subjected to in vitro erythroid differentiationand the ratio of Venus+ cells measured in the transcriptional permissiveCD71+/Ter119+ population and used for normalization of the ratio of SFFVvs. LCR-transduced cells. Venus expression in transplanted animals wascompared in erythroid cells, as this is the only population that isequally permissive for expression from both vectors. Reconstitution oftransplanted mice demonstrated a clear dominance of cells derived fromHSPC transduced with the LCR-vector, suggesting less toxicity in theHSPCs associated with erythroid lineage specific expression of theLCR-shRNAmiR (FIG. 23H). In summary, these data demonstrate that theadverse effect of BCL11A knockdown on HSC engraftment/function may becircumvented by erythroid specific miRNA expression.

LCR-Vector Mediated Erythroid Specific Knockdown of BCL11A UsingModified shRNAmiRs Yields High Levels of HbF in Human Erythroid Cells

To test the efficacy of LCR-mediated erythroid specific knockdown ofBCL11A in a human experimental system, CD34+ cells were transduced withLCR-shRNAmiR vectors containing modified shRNAmiRs 3 or 8 (FIG. 23F).The inventors first confirmed the expression profile of severalLCR-shRNAmiR vectors (LCR-shRNA*, LCRshRNAmiR3 and 8) in human cellsduring in vitro erythroid differentiation of human mPB CD34+ cells.Venus expression by the LCR-vectors and a SFFV-driven control vectorwithout shRNAmiR-cassette (SFFV-GFP) (33) was evaluated at differentstages of erythroid maturation, as defined by CD71 and GpA staining(FIG. 23I). Consistent with the findings in mouse cells shown in FIG.23G, low levels of expression were observed in CD71−/GpA− immatureerythroid cells. There was a strong upregulation of expression in CD71+single positive cells with the highest level of transgene expression inthe more mature CD71+/GpA+ double positive cells. As expected, theSFFV-GFP control drove high level constitutive expression in allsubpopulations. Following the previously described differentiationprotocol, BCL11A protein levels were measured on day 11 of culture andcompared with non-targeting and mock control vectors (LCR-shRNAmiRNT andSFFV-GFP). Significant reduction in BCL11A was observed in the cellsexpressing the modified shRNAmiR compared to the cells expressing thenon-targeting (NT) and control vector (SEW) (FIG. 24A). Gamma globinmRNA constituted 40 and 70% of total 3-like globins in cells derivedfrom CD34+ cells transduced with vectors expressing shRNAmiR3 and 8,respectively (FIG. 24B). No differences in cell growth were observedbetween cells transduced with LCR-shRNAmiRs or control vectors.Erythroid differentiation, as evaluated by surface expression of CD71,GpA and by enucleation was indistinguishable from controls (FIG. 24C),suggesting no negative impact of BCL11A knockdown upon lineage-specificexpression of the BCL11A shRNAmiRs. Strong correlation was observedbetween the levels of γ-globin mRNA (qRT-PCR) and HbF as assessed byHPLC (FIG. 24D). HbF contributed to 35% and 55% of total hemoglobin incells transduced with LCR-shRNAmiR3 and LCR-shRNAmiR8, representinglevels comparable to SFFV-promoter mediated expression (FIG. 22D andFIG. 24E). Finally, to show in proof of principle that LCR-shRNAmiRmediated knockdown allows for efficient engraftment of hCD34+ cells andinduction of γ-globin, transplantation of bone marrow derived CD34+HSPCs transduced with LCR-shRNAmiR3 or NT vectors were performed intosub-lethally irradiated NSG-mice. Due to poor development of humanerythroid cells in this xenograft model, CD34+ HSPCs were isolated fromthe bone marrow of transplanted animals 14 weeks after transplantationand subjected to erythroid differentiation in vitro. Venus+ cells wereenriched by FACS and expression of γ- and β-globin was determined byRT-PCR (FIG. 24F). Consistent with previous data, the fraction ofγ-globin of total β-globin locus output was 44.9%+5.5% for cellstransduced with LCR-shRNAmiR3, compared to ˜9%+0.5% in the two controlgroups consisting of untransduced or LCR-shRNAmiRNT transduced cells.

ShRNAs have been used extensively to analyze gene functions inbiological studies, and there is increasing interest in the use of RNAifor therapeutic purposes. BCL11A represents an attractive therapeutictarget for RNAi based modulation. BCL11A is a repressor of γ-globinexpression and thus acts as a major regulator of the fetal to adulthemoglobin switch in erythroid cells. Importantly high levels of fetalhemoglobin are associated with milder disease phenotypes in sickle celldisease (SCD) and β-thalassemias and lineage-specific knockout of BCL11Ahas been validated as a therapeutic strategy in models of SCD. In thestudies reported here, our goal was to develop a clinically applicablevector to reactivate fetal hemoglobin expression by RNAi mediatedsuppression of BCL11A. Using an optimized lentiviral vector containing amiRNA adapted shRNA (shRNAmiR) expressed from an erythroid lineagespecific pol II promoter the inventors achieved HbF levels of >50% oftotal hemoglobin in primary erythroid cells derived from transducedCD34+ HSPCs. This level of HbF induction is likely to be clinicallyeffective and compares favorably with previously published vectors (23,27, 29) utilizing pol III driven expression cassettes that lack lineagespecificity and the safety profile of SIN lentivirus vectors reportedhere.

Curative treatment for SCD can be attained with hematopoietic stem celltransplantation (HSCT). Favorable outcomes in SCD are largely dependenton the availability of matched sibling donors. Fewer than 10% of SCDpatients have unaffected HLA-matched sibling potential donors (34). Genetherapy for hemoglobinopathies offers the clear advantage of eliminatingthe risk of GVHD by the use of autologous cells. The long-term aim ofour studies is to modulate the hemoglobin switch, leading to theendogenous and physiologic induction of the protective HbF andsuppression of the sickle globin. The inventors hypothesize this dualmanipulation of expression will be the most effective therapeuticapproach to prevent toxicities in SCD including hemolysis and end organdamage of the mutant, polymerizing hemoglobin. To realize the goal oftherapeutic benefit, sufficient knockdown of BCL11A and induction of HbFon a per cell basis must occur and sufficient numbers of gene modifiedlong-lived HSC must engraft in order for chimerism of the red cellcompartment to attenuate the disease phenotype. Thus optimization ofBCL11A knockdown and preservation of reconstitution capacity oftransduced HSCP as shown here is critical to the long-term success ofgenetic therapy in SCD. As relates to the second point, the inventorsbelieve this is currently attainable, as previous data from allogeneictransplants resulting in mixed chimerism have demonstrated that as lowas 10% chimerism of the myeloid compartment is associated withperipheral blood red cell chimerism of 80-100% (35). The skewing of redcell mass after engraftment is most likely attributable to the enhancedsurvival of normal red cells compared with sickled cells. This level ofmarking of long-lived myeloid cells has recently been attained in ahuman trials utilizing lentivirus vector (36-38), including inβe-thalassemia (39).

Pol III driven shRNAs are the most commonly utilized vector systems toeffect gene knockdown by RNAi, but these vectors mediate ubiquitousexpression that may be associated with both non-specific toxicities fromhigh expression levels and sequence-specific toxicities in certain celltypes. Here the inventors demonstrate that BCL11A knockdown in HSCsimpairs engraftment of these cells in transplant settings and B celldevelopment in vivo. Although reduced engraftment in the absence ofBCL11A is an unreported phenomenon, the data reported here areconsistent with known expression of BCL11A in early HSPCs and with thereport of a -two-fold reduced HSC content in mice upon genetic deletionof BCL11A (31, 40, 41). The negative impact of BCL11A knockdown onengrafting HSCs may be more evident in the assays reported here due toincreased selective pressure present in this experimental setting.Limiting numbers of HSCs are generally present following ex vivo cultureand transduction of these cells and competition with control HSCsutilized in the assays used here may enhance the detection of toxicityat the HSC level. Within the erythroid lineage BCL11A is dispensable(24). In the data reported here, use of the erythroid specificLCR-vector, containing regulatory sequences derived from the β-globinlocus (32, 42) circumvented the negative effects of BCL11A knockdown onHSC engraftment. The LCR-vector displayed a high degree of lineagefidelity in expression of the shRNAmiR targeting BCL11A. In addition,this vector architecture has been demonstrated to reduce the risk oftransactivation of neighboring cellular genes when used to express othertransgenes (43), an important feature for clinical translation. Thus,transcriptional targeting of shRNAmiRs appears critical in the case ofBCL11A, underscoring the importance of developing effective pol II basedknockdown vectors. This approach bypasses the negative impact ofknockdown of BCL11A on HSPCs and also lymphoid cell development (30,31), avoids toxicity related to shRNA overexpression (9, 11, 19) andimproves the safety profile of the vector system, while maintaining thetherapeutic efficacy.

The use of pol II promoters for shRNA expression necessitates embeddingthe shRNA in microRNA sequences. As the majority of previously validatedeffective shRNA sequences are derived from analyses performed using polIII promoters and the majority of commercially available knockdownsystems are based on pol III promoters, conversion of shRNA sequencesinto a pol II configuration is important. In spite of significantresearch in this area, guidelines for conversion of shRNA sequencesderived from effective pol III based vectors into pol II based shRNAmiRvectors are lacking. Here by comparing the results of RNA processingfrom cells transduced with both types of vectors in parallel theinventors confirmed that different small RNA products are generated withrespect to the target matched sequences resulting in a markedly reducedefficiency of target knockdown via pol II based vectors. The matureguide strand sequences produced from pol II versus pol III systemscontaining identical target mRNA matched sequences are generally shiftedby 3-5 nt relative to each other. Addition of 3-5 U-residues from thepol III termination signal to the 3′ end of the shRNA transcript leadsto a corresponding shift of the Dicer cleavage site, proving thedominant role of the 3′-counting rule for Dicer cleavage (44, 45). Theshift of the guide strand in pol III versus pol II has a major impact onknockdown efficiency, as the seed region is altered and thethermodynamical properties and terminal nucleotide identity of the smallRNA duplex changes, thereby impacting guide strand incorporation intothe RISC-effector complex (4, 5, 46, 47). Re-engineering shRNAmiRs tomimic the mature guide strand sequences produced by pol III-drivenshRNAs led to enhanced processing and improved knockdown of the targetmRNA. This approach should be applicable for the development of vectorstargeting other genes using pol II promoters, including other lineagespecific expression cassettes.

In summary, the data demonstrate critical features of RNA processingrelevant to the use of shRNA in different vector contexts, and alsoprovide a strategy for lineage-specific gene knockdown that circumventsadverse consequences of widespread expression. The findings haveimportant implications for design of microRNA embedded shRNAs and theirapplication in RNAi based gene therapy approaches.

Example 11

Efficacy Studies of Transduction of BCL11A shRNAmiR in Health DonorHuman CD34+ Cells.

The transcriptional repressor BCL11A represents a therapeutic target forβ-hemoglobinopathies. The selectively suppression of BCL11A in erythroidcells via pol II promoter expressed microRNA adapted shRNAs (shRNAmiRs)resulted in effective knockdown of BCL11A in both murine and humancells. Expressing the modified shRNAmiRs in an erythroid-specificfashion circumvented the adverse effects on murine HSC engraftment and Bcell development (see EXAMPLE 10 supra) and led to efficient BCL11Aknockdown and high levels of HbF in primary human CD34-derived erythroidcells and in human erythroid cells differentiated in vitro after fullengraftment of modified CD34+ cells in murine xenografts. The inventorsalso demonstrated effective induction of HbF in erythroid cells derivedfrom transduced CD34 cells obtained from a donor with sickle celldisease.

In a series of experiments, GCSF mobilized CD34 from healthy donors weretransduced with a vector expressing a non-targeting shRNA (LCR-NT) orBMS11-D12G5, and subjected to erythroid in vitro differentiation.

BCL11A D12G5-2 shRNA: Sense (SEQ. ID. NO: 43)ACGCTCGCACAGAACACTCATGGATTaccatgtggtagagAATCCATGAG TGTTC TGTGCGAGAnti-sense (SEQ. ID. NO: 44)CGCACTCGCACAGAACACTCATGGATTactaccacatggagAATCCATGA GTGTT CTGTGCGA

FIG. 27A is a Western blot of in vitro differentiated erythroid cellsderived from transduced CD34 cells showing BCL11A isoforms (L and XL)and 13-ACTIN as loading control and demonstrating effective knock-downof BCL11A XL. FIG. 27B shows quantification of BCL11A knock down inerythroid cells. Data is derived from Western blots as shown in FIG.27A. Data summarizes three independent experiments using cells from asingle donor. (Error bars: SD)

FIG. 27C shows induction of gamma globin in erythroid cells as assessedby RT-qPCR and hemoglobin (HbF) assessed by HPLC.

Example 12 Quantification of BCL11A Knock Down in Erythroid Cells.

The engraftment of the transduced CD34+ cells into NSG immunodeficientmice were studied, including the effectiveness of the in vivo knockdownof the BCL11A expression. Human CD34 were transduced with LCR-NT orBMS11-D8G5 and injected into sublethally irradiated NSG-recipient mice.Bone marrow CD34+ were isolated 14 weeks later and subjected toerythroid in vitro differentiation. FIG. 28 shows induction of gammaglobin in erythroid cells as assessed by RT-qPCR.

Example 13

Knockdown of BCL11A and Induction of Fetal Hemoglobin in Erythroid CellsDerived from Transduced CD34 Cells from a Sickle Cell Patient.

Bone marrow CD34 were isolated from a SCD-patient which received HUtreatment and had high baseline HBF. The cells were transduced withLCR-NT or LCR-D12G5 and subjected to erythroid in vitro differentiation.

BCL11A knock-down was studied in sickle cell patient cells. Bone marrowCD34 were isolated from a sickle cell patient and the cells weretransduced with LCR-NT or LCR-D12G5-2 (untransduced cells used as anadditional control) and subjected to erythroid differentiation in vitro.FIG. 29A shows Western blots showing of BCL11A (L and XL isoforms) andβ-ACTIN as loading control and demonstrates effective knock-down ofBLC11A-XL. Each panel (labeled 1-6 below the lane) represents anindependent experiment using cells from a single donor. FIG. 29B showsquantification of BCL11A knockdown in erythroid cells. Data is derivedfrom Western blots shown in FIG. 29A. FIG. 29C shows resulting inductionof HbF by HPLC. This patient was receiving hydroxyurea treatment whichaccounts for the high baseline Hb F level.

Example 14 An Embodiment of a Treatment Protocol Initial Evaluation

Patients will undergo standard work-up for autologous bone marrowtransplantation according to institutional guidelines, and then undergotwo bone marrow harvests at a minimum of 4 weeks apart that will be usedfor a back-up marrow (minimum of 2×10⁶ CD34+ cells/kg) and for a harvestof autologous bone marrow for gene transfer (target of 5×10⁶ CD34+cells/kg with a minimum of 4×10⁶ CD34+ cells/kg).

Harvest of a Back-Up Autologous Graft

Hematopoietic cells will be collected from the patient in advance of thetreatment, to serve as a salvage procedure (“back-up graft”), shouldthere be no hematopoietic recovery observed 6 weeks following theinjection of genetically-manipulated cells, or should manipulated cellsfail to meet release criteria. Bone marrow (up to 20 cc/kg) will beharvested from the patient under general anesthesia from the posterioriliac crests on both sides by multiple punctures at a minimum of 4 weeksprior to gene therapy. A portion of the bone marrow containing 2×10⁶CD34+ cells/kg will be frozen and stored unmanipulated in liquidnitrogen vapors (162° C. and −180° C.) according to standard clinicalprocedures for autologous bone marrow collection to constitute theback-up graft. The remainder of the harvest will be selected for CD34+cells (described below) and utilized for gene modification (describedbelow).

Bone Marrow Harvest

The remainder of the first bone marrow harvest in excess of the neededback up marrow will be utilized with a second bone marrow harvest forgene transfer. The second harvest will occur no sooner than 4 weeksafter the initial harvest (described above). For the second harvest,bone marrow will again be harvested from the patient under generalanesthesia from the posterior iliac crests on both sites by multiplepunctures. The amount of marrow collected will be up to 20 ml/kg of bodyweight. This will give a total nucleated cell count of greater than˜4×10⁸ cells/kg. This in turn should yield a CD34+ cell dose of greaterthan 4×10⁶ cells/kg after CD34+ cell selection.

Subjects from whom the estimated CD34+ count of both harvests is <4×10⁶cells/kg will not receive conditioning. After a period of at least 6weeks, if the subject wishes to remain on study, he may be harvestedagain. If the subject does not wish to be harvested again, he will bewithdrawn from the study.

Subjects withdrawn from the study prior to administration of transducedCD34+ cells will resume normal clinical care (supportive care and/orallogeneic HSCT). Efficacy and safety assessments will not be carriedout from the point of withdrawal and data will not be collected for thedatabase.

CD34+ Cell Isolation, Pre-Stimulation, and Transduction CD34+ CellPurification.

To allow sufficient time for clearance of conditioning agents andminimize the time of pre-stimulation and culture, whole bone marrow willbe held overnight. All the manufacturing steps are performed in theConnell & O'Reilly Families Cell Manipulation Core Facility at the DFCI.The bone marrow will be red cell-depleted by density gradientcentrifugation. CD34+ cells will be positively selected from the bonemarrow mononuclear cells using the CliniMACS reagent and instrument.Quality control (QC) samples are taken to assess purity and sterility.Purified cells will be immediately processed for pre-stimulation andtransduction.

CD34+ Pre-Stimulation and Transduction

Transduction will be carried out on one or both harvests. Transductionof cells in excess of the back-up marrow target from the first harvestwill be transduced and frozen for future use. The second harvest will beused for gene transfer in its entirety and the transduced product of thesecond harvest will be infused with the thawed transduced cells from thefirst harvest after conditioning.

Purified CD34+ cells are seeded in closed culture bags at a density of0.5-1×10⁶/ml in serum-free medium supplemented with growth factors(IL-3, SCF, FLT3L, TPO) and placed in an incubator at 37° C., 5% CO₂.After 24-30 hours, cells are harvested and counted. Additional QCtesting includes cell viability, and Colony Forming Unit (CFU) assay.Cells are transferred to a new culture bag and treated with lentiviralsupernatant. For this first round of transduction, cells are incubatedfor 18-24 hours. Cells are then harvested, counted, and transferred to anew bag, with lentiviral supernatant for a second round of transduction.

Final Harvest and Formulation

After the second round of transduction, cells are harvested, washed inplasmalyte and resuspended in their final formulation (PLASMALYTE, 1%HSA) in a volume of 50-100 mL. All cells available after removal of theQC samples will be infused into the patient. QC includes cell count,viability, sterility on wash supernatant, Mycoplasma, Endotoxin onsupernatant, phenotype, CFU, RCL (samples taken and archived),insertional analysis, and average vector copy number by qPCR (culturedcells). A sample for Gram stain is taken from the product immediatelybefore delivery to the patient.

Testing Prior to Subject Re-Infusion

Samples are collected during and at the end of the procedure for cellcount and viability (trypan blue exclusion or equivalent), sterility,mycoplasma, transduction efficiency (vector copy number), Gram stain,endotoxin and RCL testing. Of these only cell viability, sterility (inprocess, 72 hours), Gram stain and endotoxin measurements will beavailable prior to infusion.

If microbiological cultures reveal transient bacterial contamination, byGram stain or positive culture at 72 hours, Cell Manipulation CoreFacility staff will contact the PI, the assistant medical director andattending physician to decide whether to infuse the back-up harvest orinfuse the product with antibiotic coverage. If back-up harvest isinfused, the subject will be withdrawn from the protocol. If the cellviability is <70%, sterility testing is positive, or endotoxin is >5EU/kg/hr, the cells will not be returned, back-up harvest will beinfused and the subject will be withdrawn from the protocol.

If viable cell count from both harvests/transductions is greater than orequal to 4×10⁶ CD34+ cells/kg at the end of transduction, cells will beinfused. If viable cell count from both harvests/transductions is lessthan 4×10⁶ CD34+ cells/kg at the end of transduction, cells will not beinfused and back-up harvest will be infused 48 hours later.

Subject Conditioning Regimen

Subjects will receive myeloablative conditioning with Busulfan (˜4 mg/kgintravenously daily, adjusted for weight, (given over 3 hours oncedaily) administered on days -4 to -2, prior to infusion of transducedcells. Conditioning will occur concurrent with purification andtransduction of bone marrow cells. Busulfan levels will be drawn on all3 days of administration, and levels on days 1 and 2 will be used toadjust the area under the curve target. Infusion of transduced cells

Cells will be infused intravenously over 30-45 minutes after standardprehydration and premedication according to Boston Children's HospitalHematopoietic Stem Cell Transplantation Unit standard guidelines. Thisstandard requires that the patient be on continuous cardiac, respiratoryand oxygen saturation monitor throughout the infusion and for 30 minutesafterwards. Vital signs will be measured and recorded pre-transfusion,15 minutes into transfusion, every hour for duration of infusion, andend of transfusion. The RN will stay with the patient for the first 5minutes of the transfusion. If two transduction products areadministered, the second transduced product will be administered withoutdelay after the first.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

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List of Synthetic miR Oligonucleotides

BCL11A miR1 oligos: Sense (SEQ ID NO: 1)ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCGagAnti-sense (SEQ ID NO: 2)CGCActCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGTGTTCTGTGCGaBCL11A miR2 oligos: Sense (SEQ ID NO: 3)ACGCTCCAGAGGATGACGATTGTTTActccatgtggtagagTAAACAATCGTCATCCTCTGGagAnti-sense (SEQ ID NO: 4)CGCActCCAGAGGATGACGATTGTTTActctaccacatggagTAAACAATCGTCATCCTCTGGaBCL11A E3 oligos:   Sense (SEQ ID NO: 5)ACGCTTCGGAGACTCCAGACAATCGCctccatgtggtagagGCGATTGTCTGGAGTCTCCGAagAnti-sense (SEQ ID NO: 6)CGCActTCGGAGACTCCAGACAATCGCctctaccacatggagGCGATTGTCTGGAGTCTCCGAaBCL11A D8 oligos: Sense (SEQ ID NO: 7)ACGCTTTCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAAagAnti-sense (SEQ ID NO: 8)CGCActTTCTCTTGCAACACGCACAGActctaccacatggagTCTGTGCGTGTTGCAAGAGAAaBCL11A XLC4 or C4 oligos: Sense (SEQ ID NO: 9)ACGCTACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGTagAnti-sense (SEQ ID NO: 10)CGCActACAGTACCCTGGAGAAACACActctaccacatggagTGTGTTTCTCCAGGGTACTGTaNon-targeting oligos: Sense (SEQ ID NO: 11)ACGCTCAACAAGATGAAGAGCACCAActccatgtggtagagTTGGTGCTCTTCATCTTGTTGagAnti-sense (SEQ ID NO: 12)CGCActCAACAAGATGAAGAGCACCAActctaccacatggagTTGGTGCTCTTCATCTTGTTGaBCL11A E3G5 or E3 mod oligos: (modified version) Sense (SEQ ID NO: 13)ACGCTGCGCTCGGAGACTCCAGACAActccatgtggtagagTTGTCTGGAGTCTCCGAGCGC agAntisense (SEQ ID NO: 14)CGCActGCGCTCGGAGACTCCAGACAActctaccacatggagTTGTCTGGAGTCTCCGAGCGC aBCL11A D8G5 or D8 mod oligos: (modified version) Sense (SEQ ID NO: 15)ACGCTGCGCTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAGCGC agAntisense (SEQ ID NO: 16)CGCActGCGCTTCTCTTGCAACACGCActctaccacatggagTGCGTGTTGCAAGAGAAGCGC aBCL11A XLC4G5 oligos: (modified version) Sense (SEQ ID NO: 17)ACGCTGCGCACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTGCGC agAntisense (SEQ ID NO: 18)CGCActGCGCACAGTACCCTGGAGAAActctaccacatggagTTTCTCCAGGGTACTGTGCGC a mIR1(SEQ ID NO: 25)CGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCG (shRNA1 or E3)(SEQ ID NO: 26)TCGGAGACTCCAGACAATCGCctccatgtggtagagGCGATTGTCTGGAGTCTCCGA (shRNA2 or B5)(SEQ ID NO: 27)CCTCCAGGCAGCTCAAAGATCctccatgtggtagagGATCTTTGAGCTGCCTGGAGG (shRNA3 or D8)(SEQ ID NO: 28)TTCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAA(shRNA4 or B11) (SEQ ID NO: 29)TCAGGACTAGGTGCAGAATGTctccatgtggtagagACATTCTGCACCTAGTCCTGA(shRNA5 or 50D12 or D12) (SEQ ID NO: 30)GATCGAGTGTTGAATAATGATctccatgtggtagagATCATTATTCAACACTCGATC(shRNA6 or 50A5 or A5) (SEQ ID NO: 31)CAGTACCCTGGAGAAACACATctccatgtggtagagATGTGTTTCTCCAGGGTACTG(shRNA7 or 50B11) (SEQ ID NO: 32)CACTGTCCACAGGAGAAGCCActccatgtggtagagTGGCTTCTCCTGTGGACAGTG(shRNA8 or 50C4) (SEQ ID NO: 33)ACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGT mIR1G5(SEQ ID NO: 34)gcgcCGCACAGAACACTCATGctccatgtggtagagCATGAGTGTTCTGTGCGgcgc(shRNA1mod or E3G5) (SEQ ID NO: 35)gcgcTCGGAGACTCCAGACAActccatgtggtagagTTGTCTGGAGTCTCCGAgcgc(shRNA2mod or B5G5) (SEQ ID NO: 36)gcgcCCTCCAGGCAGCTCAAActccatgtggtagagTTTGAGCTGCCTGGAGGgcgc(shRNA3mod or D8G5) (SEQ ID NO: 37)gcgcTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAgcgc(shRNA4mod or B11G5) (SEQ ID NO: 38)gcgcTCAGGACTAGGTGCAGActccatgtggtagagTCTGCACCTAGTCCTGAgcgc(shRNA5mod or 50D12G5 or D12G5) (SEQ ID NO: 39)gcgcGATCGAGTGTTGAATAActccatgtggtagagTTATTCAACACTCGATCgcgc(shRNA6mod or 50A5G5) (SEQ ID NO: 40)gcgcCAGTACCCTGGAGAAACctccatgtggtagagGTTTCTCCAGGGTACTGgcgc(shRNA7mod or 50B11G5) (SEQ ID NO: 41)gcgcCACTGTCCACAGGAGAActccatgtggtagagTTCTCCTGTGGACAGTGgcgc(shRNA8mod or 50C4G5 or C4G5) (SEQ ID NO: 42)gcgcACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTgcgc(BCL11A D12G5-2 shRNA):  Sense (SEQ. ID. NO: 43)ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCGAG(BCL11A D12G5-2 shRNA):  Anti-sense (SEQ. ID. NO: 44)CGCACTCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGTGTTCTGTGCGA(SEQ. ID. NO: 86)5′-CCGGCGCACAGACACTCATGGATTCTCGAGATCCATGAGTGTTCTGTGCGTTTTT-3′(SEQ. ID. NO: 87)5′CGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCGA GTG-3′(SEQ. ID. NO: 88)5′CGCTGCGCCGCACAGAACACTCATGctccatgtggtagagCATGAGTGTTCTGTGCGGCGCA GTG-3′(SEQ. ID. NO: 89)5′-CCGGACAGTACCCTGGAGAACACACTCGAGTGTGTTTCTCCAGGGTACTGTTTTTT-3′(SEQ. ID. NO: 90)5′-CGCTACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGT AGTG-3′(SEQ. ID. NO: 91)5′CGCTGCGCACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTGCGCAGT G-3′(SEQ. ID. NO: 92)5′-CCGGTTCTCTTGCACACGCACAGACTCGAGTCTGTGCGTGTTGCAGAGATTTTT-3′(SEQ. ID. NO: 93)5′CGCTTTCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAAAGT G-3′(SEQ. ID. NO: 94)5′CGCTGCGCTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAGCGCAGT G-3′(SEQ. ID. NO: 95)5′-CCGGGATCGAGTGTTGATATGATCTCGAGATCATTATTCACACTCGATCTTTTT-3′(SEQ. ID. NO: 96)5′-CGCTGATCGAGTGTTGAATAATGATctccatgtggtagagATCATTATTCAACACTCGATC AGTG-3′(SEQ. ID. NO: 97)5′CGCTGCGCGATCGAGTGTTGAATAActccatgtggtagagTTATTCAACACTCGATCGCGCAGT G-3′

New name Old name shRNA1 E3 shRNA2 B5 shRNA3 D8 shRNA4 B11 shRNA5 50D12or D12 shRNA6 50A5 or A5 shRNA7 50B11 shRNA8 50C4 shRNA1mod E3G5shRNA2mod B5G5 shRNA3mod D8G5 shRNA4mod B11G5 shRNA5mod 50D12G5 or D12G5shRNA6mod 50A5G5 shRNA7mod 50B11G5 shRNA8mod 50C4G5 or C4G5

1-80. (canceled)
 81. A lentiviral vector comprising a synthetic BCL11AmicroRNA comprising: a) a first BCL11A segment, a loop segment; and b) asecond BCL11A segment arranged in tandem in a 5′ to 3′ direction,wherein the loop segment is between and directly linked to the first andsecond BCL11A segments, wherein the first BCL11A segment starts with a-GCGC- at the 5′ end and the second BCL11A segment ends with a -GCGC- atthe 3′ end, and wherein the first BCL11A segment is complementary to thesecond BCL11A segment so that the first and second BCL11A segments basepair to form a hairpin loop with the loop segment forming the loopportion of the hairpin loop thus formed.
 82. The lentiviral vector ofclaim 81, wherein the lentiviral vector is derived from a lentivirusselected from the group consisting of: human immunodeficiency virus type1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), caprinearthritis-encephalitis virus (CAEV), equine infectious anemia virus(EIAV), feline immunodeficiency virus (FIV), bovine immune deficiencyvirus (BIV), and simian immunodeficiency virus (SIV).
 83. The lentiviralvector of claim 81, further comprising a spleen focus-forming viruspromoter, a tetracycline-inducible promoter, or a β-globin locus controlregion and a β-globin promoter.
 84. The lentiviral vector of claim 81,wherein the first and second BCL11A segments of the synthetic BCL11AmicroRNA are about 18 to 25 nucleotides long.
 85. The lentiviral vectorof claim 81, wherein the first BCL11A segment of the synthetic BCL11AmicroRNA contains a sequence derived from a BCL11A mRNA sequence. 86.The lentiviral vector of claim 81, wherein the first BCL11A segment ofthe synthetic BCL11A microRNA comprises the nucleotide sequence setforth in any one of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, 64, and 65.87. The lentiviral vector of claim 81, wherein the first BCL11A segmentof the synthetic BCL11A microRNA comprises the nucleotide sequence setforth in SEQ ID NO:
 61. 88. The lentiviral vector of claim 81, whereinthe loop segment of the synthetic BCL11A microRNA is derived from amicroRNA.
 89. The lentiviral vector of claim 81, wherein the loopsegment of the synthetic BCL11A microRNA is derived from a microRNAselected from the group consisting of: miR-142, miR-155, miR-181 andmiR-223.
 90. The lentiviral vector of claim 81, wherein the loop segmentof the synthetic BCL11A microRNA is derived from miR-223.
 91. Thelentiviral vector of claim 81, wherein the loop segment of the syntheticBCL11A microRNA comprises the polynucleotide sequence set forth in SEQID NO:
 68. 92. The lentiviral vector of claim 81, wherein the syntheticBCL11A microRNA comprises a polynucleotide sequence set forth in any oneof SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, and
 42. 93. Thelentiviral vector of claim 81, wherein the synthetic BCL11A microRNAcomprises a polynucleotide sequence set forth in SEQ ID NO:
 39. 94. Acomposition comprising the lentiviral vector of claim
 81. 95. Apharmaceutical composition comprising the lentiviral vector of claim 81and a pharmaceutically acceptable carrier or diluent.